Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1-93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid.

Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination.

Epsilon toxin (ET) is produced by Clostridium perfringens types B and D and causes severe neurological disorders in animals. ET has been observed binding to white matter, suggesting that it may target oligodendrocytes. In primary cultures containing oligodendrocytes and astrocytes, we found that ET (10(-9) M and 10(-7) M) binds to oligodendrocytes, but not to astrocytes. ET induces an increase in extracellular glutamate, and produces oscillations of intracellular Ca(2+) concentration in oligodendrocytes. These effects occurred without any change in the transmembrane resistance of oligodendrocytes, underlining that ET acts through a pore-independent mechanism. Pharmacological investigations revealed that the Ca(2+) oscillations are caused by the ET-induced rise in extracellular glutamate concentration. Indeed, the blockade of metabotropic glutamate receptors type 1 (mGluR1) prevented ET-induced Ca(2+) signals. Activation of the N-methyl-D-aspartate receptor (NMDA-R) is also involved, but to a lesser extent. Oligodendrocytes are responsible for myelinating neuronal axons. Using organotypic cultures of cerebellar slices, we found that ET induced the demyelination of Purkinje cell axons within 24 h. As this effect was suppressed by antagonizing mGluR1 and NMDA-R, demyelination is therefore caused by the initial ET-induced rise in extracellular glutamate concentration. This study reveals the novel possibility that ET can act on oligodendrocytes, thereby causing demyelination. Moreover, it suggests that for certain cell types such as oligodendrocytes, ET can act without forming pores, namely through the activation of an undefined receptor-mediated pathway.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins.

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.

Characterization of the enzymatic activity of Clostridium perfringens TpeL.

TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We confirmed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we confirmed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.

CD44 Promotes intoxication by the clostridial iota-family toxins.

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

Aqueous extract of Vitex trifolia L. (Labiatae) inhibits LPS-dependent regulation of inflammatory mediators in RAW 264.7 macrophages through inhibition of Nuclear Factor kappa B translocation and expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Vitex trifolia L. (Labiatae), a widespread tree found from the Asia-Pacific to the east Africa regions is used in the traditional medicine of the Pacific islands to treat inflammatory-associated conditions. AIM OF THE STUDY: We herein evaluated its in vitro regulatory effects on the expression profile of lipopolysaccharide (LPS)-induced inflammatory genes focusing on regulation of chemokines C-X-C motif 10 (CXCL-10) and C-C motif ligand 3 (CCL-3) and cyclo-oxygenase (COX)-2. Furthermore, the plant effect on the LPS-mediated activation of Nuclear Factor kappa B (NF-κB) was also studied. MATERIALS AND METHODS: Aqueous extract of Vitex trifolia leaves was prepared and evaluated for its effect on LPS-induced stress and toxicity-related genes in RAW 264.7 macrophage cells using RT(2) Profiler Polymerase Chain Reaction (PCR) Array System. Effects of the extract on LPS-induced chemokines CCL-3 and CXCL-10, COX-2, and NF-κB p50 and p65 mRNA levels were also studied using Reverse Transcription quantitative PCR (RT-qPCR) technique. Translocation of the nuclear factor was further assessed by measuring its nuclear p65 subunit via an ELISA-based TransAM method. RESULTS: Vitex trifolia extract at 5000µg/ml exerted a significant inhibitory effect on the expression of various LPS-induced inflammatory genes in RAW 264.7 cells after 8h of incubation time. Using RT-qPCR, this anti-inflammatory effect was further confirmed by significant inhibition of CCL-3 and CXCL-10 mRNA production in LPS-stimulated RAW 264.7 cells upon treatment with 2500µg/ml of Vitex trifolia extract. Furthermore, the inhibitory activity of this plant on LPS-induced COX-2 mRNA was also observed at a concentration of 2500µg/ml in a time-dependent manner. TransAM assays showed that LPS-induced NF-κB translocation was also inhibited by Vitex trifolia extract even at a concentration of extract as low as 250µg/ml. RT-qPCR assays showed that aqueous extract of Vitex trifolia leaves had a significant inhibitory activity on LPS-induced p50 mRNA synthesis. Interestingly, however, no effect on p65 subunit mRNA expression was observed. Moreover, PCR array analysis showed that LPS-induced inflammatory and apoptosis genes under NF-κB control are also repressed by the extract. CONCLUSION: The anti-inflammatory properties of Vitex trifolia extract seem associated with inhibition of NF-κB translocation through a reduction in the expression level of NF-κB p50 but interestingly not p65 subunit mRNA. The regulatory effects of Vitex trifolia on NF-κB and consequently on inflammation mediators such as chemokines CCL-3 and CXCL-10, and COX-2 provide new evidence of its efficacy and emphasise its high potential therapeutic value. However, further in vivo experiments are still required to validate its utilization as a remedy against inflammatory diseases.

A review of traditional remedies of ciguatera fish poisoning in the Pacific.

Ciguatera fish poisoning (CFP) is an illness caused by eating tropical coral fish contaminated with ciguatoxins (CTXs). The clinical management of patients with CFP is generally supportive and symptomatic in nature as no antidote exists. Of the many drugs prescribed, several have been claimed to be efficient in small, uncontrolled studies, but the outcomes of treatments with these medicines are often contradictory. In New Caledonia, traditional remedies are commonly employed in the treatment of CFP and of the 90 plant species catalogued as useful in CFP, the most popular herbal remedy by far is a decoction prepared from the leaves of Heliotropium foertherianum Diane & Hilger (Boraginaceae). Other important plants used in the treatment of CFP include Euphorbia hirta L. (Euphorbiaceae) and Vitex L. sp. (Lamiaceae). This review focuses on the evidence for efficacy of these species and pharmacological studies which support their use. Other plants used in CFP and the conventional treatment of CFP are also discussed briefly.

Detection of ciguatoxin-like and paralysing toxins in Trichodesmium spp. from New Caledonia lagoon.

Marine pelagic cyanobacteria Trichodesmium are widespread in the New Caledonia lagoon. Blooms of these Oscillatoriales are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain certain types of paralysing toxins. In the present study, toxicity experiments were conducted on lipid- and water-soluble extracts of freeze-dried samples of these cyanobacteria. Lipid-soluble fractions revealed a ciguatoxin-like activity in both in vivo (mouse bioassay) and in vitro (mouse neuroblastoma cells assay and receptor binding assay using tritiated brevetoxin-3) assays. The water-soluble fractions tested on mice exhibited neurotoxicity with paralytic symptoms. These toxicities have also been observed with benthic filamentous cyanobacteria within the Oscillatoriales order, also collected in New Caledonia. This study provides an unprecedented evidence of the toxicity of Trichodesmium species from the New Caledonia lagoon. This survey also demonstrates the possible role of these cyanobacteria in ciguatera fish poisoning.

Update on methodologies available for ciguatoxin determination: perspectives to confront the onset of ciguatera fish poisoning in Europe.

Ciguatera fish poisoning (CFP) occurs mainly when humans ingest finfish contaminated with ciguatoxins (CTXs). The complexity and variability of such toxins have made it difficult to develop reliable methods to routinely monitor CFP with specificity and sensitivity. This review aims to describe the methodologies available for CTX detection, including those based on the toxicological, biochemical, chemical, and pharmaceutical properties of CTXs. Selecting any of these methodological approaches for routine monitoring of ciguatera may be dependent upon the applicability of the method. However, identifying a reference validation method for CTXs is a critical and urgent issue, and is dependent upon the availability of certified CTX standards and the coordinated action of laboratories. Reports of CFP cases in European hospitals have been described in several countries, and are mostly due to travel to CFP endemic areas. Additionally, the recent detection of the CTX-producing tropical genus Gambierdiscus in the eastern Atlantic Ocean of the northern hemisphere and in the Mediterranean Sea, as well as the confirmation of CFP in the Canary Islands and possibly in Madeira, constitute other reasons to study the onset of CFP in Europe [1]. The question of the possible contribution of climate change to the distribution of toxin-producing microalgae and ciguateric fish is raised. The impact of ciguatera onset on European Union (EU) policies will be discussed with respect to EU regulations on marine toxins in seafood. Critical analysis and availability of methodologies for CTX determination is required for a rapid response to suspected CFP cases and to conduct sound CFP risk analysis.

Prevalence, symptoms and chronicity of ciguatera in New Caledonia: results from an adult population survey conducted in Noumea during 2005.

Ciguatera is a widespread ichthyosarcotoxism which causes gastrointestinal, neurological and cardiovascular disturbances. Investigations conducted by ORSTOM in 1992 highlighted a prevalence of 25% in the adult population of Noumea, New Caledonia. The main objective of our study was to estimate the prevalence of ciguatera and the persistence of symptoms by sex and by ethnicity among adult patients of a nurse clinic in Noumea in 2005. Investigations were conducted from 1st January to 15th June 2005. During this period, 559 patients were included: 165 males and 394 females. Among them, 37.8% were poisoned at least once in their life. This rate was independent of gender and ethnicity, but was significantly higher in age groups above 40 years. Neurological signs were more frequent (>80%) than gastrointestinal (<50%) and cardiac signs (<15%). Symptoms presented no difference between ethnic or gender groups, even for subjective signs. Most of poisonings were due to carnivorous fishes, but quite all species living in the lagoon were quoted. Symptoms persisted more than one year for 34% of the population, in both Melanesians and Caucasians. This study shows a significant increase of ciguatera prevalence, and its chronicity for 1/5 of European cases.

First identification of the neurotoxin homoanatoxin-a from mats of Hydrocoleum lyngbyaceum (marine cyanobacterium) possibly linked to giant clam poisoning in New Caledonia.

We report the first identification of homoanatoxin-a from benthic marine cyanobacteria (Hydrocoleum lyngbyaceum) samples collected in Lifou (Loyalty Islands, New Caledonia), where cases of giant clams (Tridacna maxima) intoxications were recorded during a severe ciguatera fish poisoning outbreak. Homoanatoxin-a was also detected in extracts of giant clams harvested in the surroundings of the contaminated area suggesting the possible link between these poisoning events and the occurrence of potentially neurotoxic Hydrocoleum.

Marine toxic cyanobacteria: diversity, environmental responses and hazards.

Toxic cyanobacterial blooms have been a primary concern predominantly in the plankton of freshwater bodies. Recently, however, the toxicity of benthic cyanobacteria is increasingly attracting attention of the scientific community and environmental agencies. The occurrence of toxic strains in benthic cyanobacteria is intimately linked to our understanding of the diversity and ecological responses of these organisms under field conditions. To that effect, we are engaged in combined morphotypic and genotypic characterization (polyphasic) of benthic natural populations of cyanobacteria in tropical lagoons and coral reefs, with the objective to provide a reliable reference for further comparative work. The methods of identification based on phenotypic properties and those based on molecular tools for genotypic identification are correlated. The approach is based on identifying the occurrences of cyanobacterial benthic blooms, tested for purity and analyzed by application of molecular tools. The questions addressed include the distinction between marine and freshwater taxa, between populations in geographically separate regions as well as between their potential vs. expressed toxicity.

Ciguatera risk management in French Polynesia: the case study of Raivavae Island (Australes Archipelago).

Based on epidemiological data available through long-term monitoring surveys conducted by both the Public Health Directorate and the Louis Malardé Institute, ciguatera is highly endemic in French Polynesia, most notably in Raivavae (Australes) which appears as a hot spot of ciguatera with an average incidence rate of 140 cases/10,000 population for the period 2007-2008. In order to document the ciguatera risk associated with Raivavae lagoon, algal and toxin-based field monitoring programs were conducted in this island from April 2007 to May 2008. Practically, the distribution, abundance and toxicity of Gambierdiscus populations, along with the toxicity levels in 160 fish distributed within 25 distinct species, were assessed in various sampling locations. Herbivores such as Scarids (parrotfish) and Acanthurids (unicornfish) were rated as high-risk species based on receptor-binding assay toxicity data. A map of the risk stratification within the Raivavae lagoon was also produced, which indicates that locations where both natural and man-made disturbances have occurred remained the most susceptible to CFP incidents. Our findings also suggest that, locally, the traditional knowledge about ciguatera may not be scientifically complete but is functionally correct. Community education resulted in self-regulating behaviour towards avoidance of high-risk fish species and fishing locations.

Pacific ciguatoxin 1B-induced modulation of inflammatory mediators in a murine macrophage cell line.

Ciguatoxins, potent marine neurotoxins responsible for ciguatera, exert their numerous damaging effects through primary binding to the voltage-sensitive sodium channels of excitable cells. Using RAW 264.7 murine macrophages, we report the first experimental study presenting evidence that P-CTX-1B (the most potent congener from the Pacific) could modulate mRNA expression of pro- and anti-inflammatory cytokines as well as of inducible nitric oxide synthase (iNOS). P-CTX-1B, unlike other less potent marine polyether toxins, P-CTX-3C and PbTx-3, induced the overexpression of interleukin (IL)-1beta, IL-6, IL-10, tumor necrosis factor-alpha and iNOS with different magnitude and kinetic profiles, as compared to bacterial lipopolysaccharide (LPS). Unlike LPS, P-CTX-1B did not modulate IL-11 expression. In this report, we provide new evidence of the P-CTX-1B iNOS- and cytokines-inducing ability and shed new light on host response to potent neurotoxins.

Growth and toxin production in the ciguatera-causing dinoflagellate Gambierdiscus polynesiensis (Dinophyceae) in culture.

The growth and toxin production in a clonal strain of Gambierdiscus polynesiensis, TB-92, was examined in batch culture conditions. The mean growth rate at exponential phase was (0.13+/-0.03)division day(-1). Regardless of the age of cultures, all mice injected with dichloromethanolic and methanolic extracts showed symptoms specific to ciguatoxin (CTX) and maitotoxin (MTX) bioactivity, respectively. The highest total toxicity assessed in TB-92 cultures was 10.4 x 10(-4) mouse unit cell(-1). The toxin production pattern reveals an enhanced cellular toxin content with the age of the culture. CTX- and MTX-like compounds each accounted for approx. 50% of the total toxicity of TB-92 cultures, except in aged cells where CTXs were dominant. The high ciguatoxic activity of TB-92 was further confirmed in dichloromethanolic extracts by means of the receptor-binding assay. The highest CTX level monitored at late stationary phase was (11.9+/-0.4)pg P-CTX-3C equiv cell(-1). Further HPLC and LC-MS analysis revealed the presence of five CTXs congeners in lipid-soluble extracts, i.e. CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C, and of new CTX congeners. Toxin composition comparison between two G. polynesiensis strains suggests that the toxin profile is a stable characteristic in this species. G. polynesiensis clones also proved inherently more toxic than other Gambierdiscus species isolated from other geographical areas.

Characterisation of the anti-inflammatory potential of Vitex trifolia L. (Labiatae), a multipurpose plant of the Pacific traditional medicine.

AIM OF THE STUDY: Vitex trifolia L. (Labiatae) is a plant commonly employed against Ciguatera Fish Poisoning (CFP) in the Pacific region. Here, the anti-inflammatory potential of an aqueous extract of Vitex trifolia leaves was evaluated by monitoring its effects on the modulation of cytokines, the mediators of inflammation, as well as on the expression profiles of inducible nitric oxide synthase (iNOS) which produces the free radical nitric oxide (NO). MATERIALS AND METHODS: We prepared an aqueous extract from Vitex trifolia leaves and evaluated its anti-inflammatory potency by monitoring its effect on the lipopolysaccharide (LPS)-induced cytokines and iNOS mRNA over-production in RAW 264.7 macrophages using quantitative Polymerase Chain Reaction (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) methods. RESULTS: Aqueous extract of Vitex trifolia leaves showed significant dose- and time-dependent inhibitory activity on interleukin (IL)-1 beta, IL-6 and iNOS mRNA synthesis, but slight effect on tumor necrosis factor (TNF)-alpha, all of which are involved in the inflammatory response. Moreover, the plant extract seemed to induce the LPS-dependent IL-10 anti-inflammatory cytokine. These results were further confirmed by ELISA using specific antibodies to mouse IL-6, IL-10 and TNF-alpha. CONCLUSION: The anti-inflammatory effects of Vitex trifolia could validate its utilization as a traditional remedy against CFP and emphasises its potential therapeutic value against other inflammatory diseases. Therefore, this plant is a promising candidate for further screening of its active compounds through activity-guided fractionation.