This study investigated for the first time the effects of the cis isomer of resveratrol (c-RESV) on the responses of inflammatory murine peritoneal macrophages, namely on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during the respiratory burst; on the biosynthesis of other mediators of inflammation such prostaglandins; and on the expression of inflammatory genes such as inducible nitric oxide synthase (NOS)-2 and inducible cyclooxygenase (COX)-2. Treatment with 1-100 microM c-RESV significantly inhibited intracellular and extracellular ROS production, and c-RESV at 10-100 microM significantly reduced RNS production. c-RESV at 1-100 microM was ineffective for scavenging superoxide radicals (O(2)(.-)), generated enzymatically by a hypoxanthine (HX)/xanthine oxidase (XO) system and/or for inhibiting XO activity. However, c-RESV at 10-100 microM decreased nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase activity in macrophage homogenates. c-RESV at 100 microM decreased NOS-2 and COX-2 mRNA levels in lipopolysaccharide (LPS) interferon gamma (IFN-gamma)-treated macrophages. At 10-100 microM, c-RESV also significantly inhibited NOS-2 and COX-2 protein synthesis and decreased prostaglandin E(2) (PGE(2)) production. These results indicate that c-RESV at micromolar concentrations significantly attenuates several components of the macrophage response to proinflammatory stimuli (notably, production of O(2)(.-)(-) and of the proinflammatory mediators NO(.-) and PGE(2)).

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Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

This study investigated the in vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate (EGCG) on the histiophagous ciliate Philasterides dicentrarchi, which causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus L. Of the 3 polyphenols, resveratrol showed strongest antiprotozoal activity, reducing ciliate density after 1 wk culture by, on average, 91% at 50 µM, and 96% at 500 µM. EGCG reduced ciliate density by, on average, 93% at 500 µM, with no significant effect at 50 µM. Mangiferin reduced ciliate density by, on average, 56% at 500 µM, again with no significant effect at 50 µM. In view of these findings, we discuss the potential utility of chemotherapy with polyphenols as a strategy for the control of scuticociliatosis in farmed turbot. KEY WORDS: Polyphenols · Philasterides dicentrarchi · Resveratrol · Mangiferin · Epigallocatechin Resale or republication not permitted without written consent of the publisherDis Aquat Org 59: [171][172][173][174] 2004 toalexin (phytoestrogen) synthesized by Vitis vinifera in response to injury or fungal attack, and is present at high concentrations in grape skin and red wine. MATERIALS AND METHODSThe scuticociliate Philasterides dicentrarchi was obtained from ascitic fluid from the body cavity of naturally infected turbot Scophthalmus maximus, and maintained in complete L-15 medium (Leibovitz, Sigma) under the culture conditions previously described , which maintain the capacity of the parasite to infect turbot (Iglesias et al. 2002). The polyphenols trans-resveratrol (RESV), mangiferin (MA) and (-)-epigallocatechin-3-gallate (EGCG) (Fig. 1) were purchased from Sigma (Sigma Química) and maintained as 100 mM stock solutions in dimethyl sulfoxide (DMSO) at -20°C in the dark until use. To investigate antiprotozoal effects, the polyphenols (RESV, MA, or EGCG) in 1 ml of complete L-15 medium were added to the wells of 24-well sterile culture plates (Corning) containing 5 × 10 3 ciliates per well, to a final concentration of 50 or 500 µM, with subsequent incubation for 1 wk at 16°C. After this incubation period, the number of ciliates present was quantified in a 25 µl aliquot from each well: the ciliates were first inactivated with glutaraldehyde (final concentration 0.25%) for 15 min at room temperature, then counted in a hemocytometer (Iglesias et al. 2002). All experiments were done in quintuplicate. To rule out possible effects of the solvent DMSO, quintuplicate wells with L-15 medium containing the highest DMSO concentration used (0.5%) were also included. The results are expressed as means ± standard error (SEM), and statistical significances were assessed by 1-way ANOVA followed by TukeyKramer tests for multiple comparisons. RESULTS AND DISCUSSIONThe effects of RESV, MA, and EGCG (50 or 500 µM) on the Philasterides dicentrarchi trophozoite count in culture after 1 wk incubation are summarized in Fig. 2. As can be seen, the solvent DMSO had no significant effect; in previous studies we have found that DMSO at up...

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