Assessing hydroxylated polycyclic aromatic hydrocarbon (OHPAH) metabolites in bile of English sole (Parophrys vetulus) from Puget Sound, WA, USA by liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are widely monitored in marine biota from urbanized areas, due to their toxicity to aquatic organisms. Teleost fish can quickly metabolize PAHs into hydroxylated forms (OHPAHs) that, in some cases, are more toxic than the parent (unmetabolized) PAHs. But due to this fast metabolism, monitoring traditional parent PAHs in fish can cause underestimation on assessing PAH exposure. In addition, environmental levels of individual OHPAH metabolites are lacking in the literature worldwide. Therefore, we developed a rapid and accurate analytical method in which a number of individual OHPAHs metabolites are measured simultaneously in fish bile, via liquid chromatography coupled with tandem mass spectrometry, including low and high molecular weight mono- and diol-OHPAHs. We analyzed bile samples of 119 English sole (Parophrys vetulus) collected from 14 Puget Sound, WA, USA, sites, which has multiple sources of PAHs, including urban stormwater runoff, wastewater effluents, as well as an inactive creosote facility. The mean (± SD) biliary summed OHPAH (∑OHPAH) concentrations determined in English sole from urban, near-urban, and non-urban sites were 790 ± 1400 (n = 46), 310 ± 330 (n = 44) and 130 ± 200 (n = 29) ng/mL, respectively, with a maximum reaching 9400 ng/mL in a sample from an urban site. We compared these novel biliary OHPAH metabolite data with parent PAHs measured in stomach content of the same individual sole. Biliary ∑OHPAH concentrations were significantly correlated with the levels of ∑PAH in stomach content, however, with major differences in their distribution. We also demonstrated that biliary OHPAH metabolite data in English sole can potentially be used to distinguish different sampling sites due to a specific variety and intensity of PAH sources in the aquatic environment, which makes this a very important analytical approach for assessing PAH exposure in the environment.

Highly contaminated river otters (Lontra canadensis) are effective biomonitors of environmental pollutant exposure.

River otters (Lontra canadensis) are apex predators that bioaccumulate contaminants via their diet, potentially serving as biomonitors of watershed health. They reside throughout the Green-Duwamish River, WA (USA), a watershed encompassing an extreme urbanization gradient, including a US Superfund site slated for a 17-year remediation. The objectives of this study were to document baseline contaminant levels in river otters, assess otters' utility as top trophic-level biomonitors of contaminant exposure, and evaluate the potential for health impacts on this species. We measured a suite of contaminants of concern, lipid content, nitrogen stable isotopes (δ15N), and microsatellite DNA markers in 69 otter scat samples collected from twelve sites. Landcover characteristics were used to group sampling sites into industrial (Superfund site), suburban, and rural development zones. Concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ether flame-retardants (PBDEs), dichlorodiphenyl-trichloroethane and its metabolites (DDTs), and polycyclic aromatic hydrocarbons (PAHs) increased significantly with increasing urbanization, and were best predicted by models that included development zone, suggesting that river otters are effective biomonitors, as defined in this study. Diet also played an important role, with lipid content, δ15N or both included in all best models. We recommend river otter scat be included in evaluating restoration efforts in this Superfund site, and as a potentially useful monitoring tool wherever otters are found. We also report ΣPCB and ΣPAH exposures among the highest published for wild river otters, with almost 70% of samples in the Superfund site exceeding established levels of concern.

Helminth antigens modulate human PBMCs, attenuating disease progression in a humanised mouse model of graft versus host disease.

Fasciola hepatica is a trematode worm that causes fascioliasis, a neglected tropical disease in humans and livestock. To gain insight into the host-parasite interactions that facilitate infection, we have investigated the immunomodulatory properties of the parasite's tegumental coat (FhTeg), a major antigen source that is sloughed off and renewed every 2-3 h as the worm migrates through host tissue. Using mouse models of infection, we have previously shown that FhTeg induces a novel phenotype of dendritic cells that induce anergic CD4+ T-cells. We proposed that this induced state of hyporesponsiveness characterised by suppression of cell proliferation and cytokine secretion was one mechanism by which F. hepatica prevented host protective immunity to support the parasite survival. To determine if the same mechanisms are utilised during human infections, we have now examined the interaction of FhTeg with human PBMCs. FhTeg binds to and modulates cytokine production in human PBMCs, in particular targeting the CD4+ population resulting in reduced levels of TNF, IL-2 and IFNγ and increased markers of anergy. Furthermore, the adoptive transfer of FhTeg stimulated PBMCs to a humanised model of acute graft versus host disease (GvHD) attenuated disease progression by increasing survival and reducing pathological scores. These mice also displayed a significant decrease in the total number of human CD4+ cells expressing TNF, IL-2 and IFNγ in the spleen, liver and lung. This study therefore concurs with evidence from ruminant and murine models of infection suggesting that anergic CD4+ T cells are associated with successful Fasciola hepatica infection and highlights an important role for FhTeg in contributing to the overall immunosuppressive effects of this parasite.

Fasciola hepatica Extracellular Vesicles isolated from excretory-secretory products using a gravity flow method modulate dendritic cell phenotype and activity.

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.

Concentrations and profiles of organochlorine contaminants in North Pacific resident and transient killer whale (Orcinus orca) populations.

Organochlorine (OC) profiles have been used as chemical "fingerprints" to infer an animal's foraging area. North Pacific killer whale (Orcinus orca) populations are exposed to different levels and patterns of OCs based on their prey, distribution, and amount of time spent in a particular area. To characterize concentrations and profiles of OCs found in various populations of North Pacific killer whales, polychlorinated biphenyls (PCBs), including dioxin-like congeners, DDTs, and hexachlorobenzene (HCB), were measured in biopsy blubber samples of photo-identified resident (fish-eating) and transient (mammal-eating) killer whales collected from 1994 through 2002 from Russian Far East waters to the waters of the west coast of the United States, representing 10 populations. We compared blubber OC concentrations based on ecotype (resident vs. transient), sex and reproductive maturity, and geographic area. We also examined OC mixtures to determine if we could detect segregated geographical areas (foraging areas) among the six populations with sufficient sample sizes. Transients had significantly higher OC concentrations than residents and adult male whales had consistently higher OC levels compared to adult females, regardless of ecotype. Our OC profile findings indicate segregated foraging areas for the North Pacific killer whales, consistent with observations of their geographic distributions. Several potential health risks have also been associated with exposure to high levels of contaminants in top-level predators including reproductive impairment, immune suppression, skeletal deformities, and carcinoma. The results of this baseline study provide information on the geographic distribution of OCs found in North Pacific killer whales, results which are crucial for assessing the potential health risks associated with OC exposure in this species.

Chemical tracers guide identification of the location and source of persistent organic pollutants in juvenile Chinook salmon (Oncorhynchus tshawytscha), migrating seaward through an estuary with multiple contaminant inputs.

Understanding the spatial extent, magnitude, and source of contaminant exposure in biota is necessary to formulate appropriate conservation measures to reduce or remediate contaminant exposure. However, obtaining such information for migratory animals is challenging. Juvenile Chinook salmon (Oncorhynchus tshawytscha), a threatened species throughout the US Pacific Northwest, are exposed to persistent organic pollutants (POPs), including polybrominated diphenyl ether (PBDE) flame retardants and polychlorinated biphenyls (PCBs), in many developed rivers and estuaries. This study used three types of complementary chemical tracer data (contaminant concentrations, POP fingerprints, and stable isotopes), to determine the location and source of contaminant exposure for natural- and hatchery-origin Chinook salmon migrating seaward through a developed watershed with multiple contaminant sources. Concentration data revealed that salmon were exposed to and accumulated predominantly PBDEs and PCBs in the lower mainstem region of the river, with higher PBDEs in natural- than hatchery-origin fish but similar PCBs in both groups, associated with differences in contaminant inputs and/or habitat use. The POP fingerprints of the natural-origin-fish captured from this region were also distinct from other region and origin sample groups, with much higher proportions of PBDEs in the total POP concentration, indicating a different contaminant source or habitat use than the hatchery-origin fish. Stable isotopes, independent tracers of food sources and habitat use, revealed that natural-origin fish from this region also had depleted δ15N signatures compared to other sample groups, associated with exposure to nutrient-rich wastewater. The PBDE-enhanced POP fingerprints in these salmon were correlated with the degree of depletion in nitrogen stable isotopes of the fish, suggesting a common wastewater source for both the PBDEs and the nitrogen. Identification of the location and source of contaminant exposure allows environmental managers to establish conservation measures to control contaminant inputs, necessary steps to improve the health of Chinook salmon and enhance their marine survival.

Toxic Burdens of Freshwater Biofilms and Use as a Source Tracking Tool in Rivers and Streams.

Biofilms, composed of periphyton, bacteria, and organic detritus, are the base of the food web in many streams and rivers. This media adsorbs and actively sequesters organic and inorganic contaminants from the water column. Here, we demonstrate the utility of using the contaminant concentrations in the biofilm matrix as an environmental media in source tracking and understanding biological impacts at higher trophic levels. Physical partitioning of polychlorinated biphenyl (PCB) and polybrominated diphenyl ether congeners is the dominant mode of uptake from water to biofilm and bioaccumulation factor: log Kow relationships suggest that PCB uptake is often near equilibrium between log Kow 5-7. We show that the concentrations of metals in biofilms are more effective at delineating and recording spatial and temporal differences in metal inputs than bed sediments and water samples. The burden of metals in the biofilm matrix explained adverse impacts and variability in periphyton metrics and ecological integrity in macroinvertebrates. This work provides new insights into the partitioning of organic chemicals onto biofilms and shows clear linkages between metals in the biofilm matrix and ecological health of invertebrates that depend on biofilms as a food source.

Polycyclic aromatic hydrocarbons in Pacific herring (Clupea pallasii) embryos exposed to creosote-treated pilings during a piling-removal project in a nearshore marine habitat of Puget Sound.

We used manually spawned, field-deployed embryos of a common marine fish species, Pacific herring (Clupea pallasii), to evaluate accumulation of polycyclic aromatic hydrocarbons (PAHs) associated with an incomplete creosote-treated piling (CTP) removal project. Embryos near undisturbed 100-year-old CTPs (before removal) accumulated higher PAHs and exhibited higher cyp1a gene expression than embryos from reference areas. Embryos incubated close to CTP debris after CTP removal showed PAHs 90 times higher than reference areas up to a year after CTP removal. cyp1a fold-induction correlated with total embryo PAHs in all three years. Patterns of individual PAH chemicals differed slightly between embryos, wood sampled from CTPs, and passive samplers. This study illustrates the importance of using appropriate techniques and procedures to remove CTPs in aquatic environments to prevent release of toxic chemicals. Of particular concern is that incomplete CTP removal could expose sensitive life stages of fishes to chemicals that may reduce their survival.

In silico analyses of protein glycosylating genes in the helminth Fasciola hepatica (liver fluke) predict protein-linked glycan simplicity and reveal temporally-dynamic expression profiles.

Glycoproteins secreted by helminth parasites are immunogenic and represent appealing components of vaccine preparations. Our poor knowledge of the pathways that mediate protein glycosylation in parasitic flatworms hinders our understanding of how proteins are synthesised and modified, and our ability to target these pathways for parasite control. Here we provide the first detailed description of genes associated with protein glycosylation in a parasitic flatworm, focusing on the genome of the liver fluke (Fasciola hepatica), which is a globally important trematode parasite of humans and their livestock. Using 190 human sequences as search queries against currently available F. hepatica genomes, we identified 149 orthologues with putative roles in sugar uptake or nucleotide sugar synthesis, and an array of glycosyltransferase and glycosidase activities required for protein N- and O-glycosylation. We found appreciable duplication within these orthologues, describing just 87 non-redundant genes when paralogues were excluded. F. hepatica lacks many of the enzymes required to produce complex N- and O-linked glycans, which explains the genomic basis for the structurally simple glycans described by F. hepatica glycomic datasets, and predicts pervasive structural simplicity in the wider glycome. These data provide a foundation for functional genomic interrogation of these pathways with the view towards novel parasite intervention strategies.

Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host.

The parasite Fasciola hepatica infects a broad range of mammals with impunity. Following ingestion of parasites (metacercariae) by the host, newly excysted juveniles (NEJ) emerge from their cysts, rapidly penetrate the duodenal wall and migrate to the liver. Successful infection takes just a few hours and involves negotiating hurdles presented by host macromolecules, tissues and micro-environments, as well as the immune system. Here, transcriptome and proteome analysis of ex vivo F. hepatica metacercariae and NEJ reveal the rapidity and multitude of metabolic and developmental alterations that take place in order for the parasite to establish infection. We found that metacercariae despite being encased in a cyst are metabolically active, and primed for infection. Following excystment, NEJ expend vital energy stores and rapidly adjust their metabolic pathways to cope with their new and increasingly anaerobic environment. Temperature increases induce neoblast proliferation and the remarkable up-regulation of genes associated with growth and development. Cysteine proteases synthesized by gastrodermal cells are secreted to facilitate invasion and tissue degradation, and tegumental transporters, such as aquaporins, are varied to deal with osmotic/salinity changes. Major proteins of the total NEJ secretome include proteases, protease inhibitors and anti-oxidants, and an array of immunomodulators that likely disarm host innate immune effector cells. Thus, the challenges of infection by F. hepatica parasites are met by rapid metabolic and physiological adjustments that expedite tissue invasion and immune evasion; these changes facilitate parasite growth, development and maturation. Our molecular analysis of the critical processes involved in host invasion has identified key targets for future drug and vaccine strategies directed at preventing parasite infection.

Time Trends of Persistent Organic Pollutants in Benthic and Pelagic Indicator Fishes from Puget Sound, Washington, USA.

We modeled temporal trends in polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and dichlorodiphenyltrichloroethane and its metabolites (DDTs) in two indicator fish species representing benthic and pelagic habitats in Puget Sound, Washington, USA. English sole (Parophrys vetulus, benthic) index sites and larger-scale Pacific herring (Clupea pallasii, pelagic) foraging areas represented a wide range of possible contamination conditions, with sampling locations situated adjacent to watersheds exhibiting high, medium and low development. Consistency in analytical data throughout the study was maintained by either calculating method-bias-correction factors on paired samples as methods evolved or by analyzing older archived samples by current methods. PCBs declined moderately in two herring stocks from a low-development basin (2.3 and 4.0% annual rate of decline) and showed no change in the highly developed and moderately developed basins during a 16- to 21-year period. PCBs increased in English sole from four of ten sites (2.9-7.1%), and the remaining six exhibited no significant change. PBDEs and DDTs declined significantly in all herring stocks (4.2-8.1%), although analytical challenges warrant caution in interpreting DDT results. PBDEs declined in English sole from two high-development and one low-development site (3.7-7.2%) and remained unchanged in the remaining seven. DDTs increased in English sole from one high-development site (Tacoma City Waterway) and declined in two high-development and one low development site. As with herring, analytical challenges warrant caution in interpreting the English sole DDT results. It is likely that source controls and mitigation efforts have contributed to the declines in PBDEs and DDTs overall, whereas PCBs appear to have persisted, especially in the pelagic food web, despite bans in PCB production and use.

Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor.

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica's tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg's binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg's ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host's immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.

Fasciola hepatica tegumental antigens induce anergic-like T cells via dendritic cells in a mannose receptor-dependent manner.

FoxP3(+) Treg cells and anergic T cells are the two regulatory phenotypes of T-cell responses associated with helminth infection. Here, we examine the T-cell responses in mice during Fasciola hepatica infection, and to its tegumental coat antigens (FhTeg) that are shed from the fluke every 2-3 h. FhTeg comprises a rich source of glycoproteins, mainly oligomannose N-glycans that bind to mannose receptor. This study demonstrated a novel mechanism for the T-cell unresponsiveness observed during F. hepatica infection and after injection with FhTeg. Markers of T-cell anergy, such as GRAIL, EGR2, ICOS, and ITCH, are enhanced amongst CD4(+) T-cell populations during infection and following FhTeg injection. This is characterized by a lack of cytokine responses and reduced proliferative activity, which can be reversed with the addition of IL-2. FhTeg-activated dendritic cells (DCs) suppress T cells in vitro as measured by enhanced GRAIL and CTLA4 by RNA and suppressed cytokine expression in anti-CD3 stimulated CD4(+) T cells. FhTeg-treated DCs have enhanced MR expression, which is critical for DC-CD4(+) T-cell communication. Taken together, this study presents markers of anergy in a mouse model of F. hepatica infection, and improves our understanding of host-pathogen interactions and how helminths modulate host immunity.

Fasciola hepatica vaccine: we may not be there yet but we're on the right road.

Major advances have been made in identifying potential vaccine molecules for the control of fasciolosis in livestock but we have yet to reach the level of efficacy required for commercialisation. The pathogenesis of fasciolosis is associated with liver damage that is inflicted by migrating and feeding immature flukes as well as host inflammatory immune responses to parasite-secreted molecules and tissue damage alarm signals. Immune suppression/modulation by the parasites prevents the development of protective immune responses as evidenced by the lack of immunity observed in naturally and experimentally infected animals. In our opinion, future efforts need to focus on understanding how parasites invade and penetrate the tissues of their hosts and how they potentiate and control the ensuing immune responses, particularly in the first days of infection. Emerging 'omics' data employed in an unbiased approach are helping us understand liver fluke biology and, in parallel with new immunological data, to identify molecules that are essential to parasite development and accessible to vaccine-induced immune responses.

An evaluation of background levels and sources of polycyclic aromatic hydrocarbons in naturally spawned embryos of Pacific herring (Clupea pallasii) from Puget Sound, Washington, USA.

Pacific herring embryos spawned in nearshore habitats may be exposed to toxic contaminants as they develop, from exogenous sources in spawning habitats and from maternal transfer. Determining baseline concentrations of these toxic contaminants is important for evaluating the health of this species, especially during this sensitive life stage. In this study we compared concentrations of polycyclic aromatic hydrocarbons, or PAHs, in naturally spawned herring embryos from five spawning areas across Puget Sound. The summed values of 31 PAH analytes (Σ31PAH) in early- to late-stage development embryos ranged from 1.1 to 140 ng/g, wet weight. Σ31PAH concentrations increased with development time in embryos from one spawning area where the greatest concentrations were observed, and the relative abundance of PAH chemicals in late-stage embryos was similar to those in nearby sediments, suggesting accumulation from local environmental sources. PAHs in both sediments and late-stage embryos appeared to exhibit a pyrogenic pattern. Although maternal transfer of PAHs appeared to be a negligible source to embryos in spawning areas with the greatest embryo PAH concentrations, maternal transfer may have been the dominant source in embryos from spawning areas where the lowest levels of embryo-PAHs occurred. Chronic embryo mortality has been reported in spawning habitats where we observed the greatest concentration of PAHs in embryos, and necrotic tissue in herring embryos from one such location was similar in description to phototoxic PAH necrosis reported elsewhere for embryonic zebrafish.

Mannose receptor and macrophage galactose-type lectin are involved in Bordetella pertussis mast cell interaction.

Mast cells are crucial in the development of immunity against Bordetella pertussis, and the function of TLRs in this process has been investigated. Here, the interaction between mast cells and B. pertussis with an emphasis on the role of CLRs is examined. In this study, two CLRs, MGL and MR, were detected for the first time on the surface of mast cells. The involvement of MR and MGL in the stimulation of mast cells by heat-inactivated BP was investigated by the use of blocking antibodies and specific carbohydrate ligands. The cell wall LOS of BP was also isolated to explore its role in this interaction. Mast cells stimulated with heat-inactivated BP or BP LOS induced TNF-α, IL-6, and IFN-γ secretion, which was suppressed by blocking MR or MGL. Inhibition of CLRs signaling during BP stimulation affected the ability of mast cells to promote cytokine secretion in T cells but had no effect on the cell-surface expression of ICAM1. Blocking MR or MGL suppressed BP-induced NF-κB expression but not ERK phosphorylation. Syk was involved in the CLR-mediated activation of mast cells by BP. Bacterial recognition by immune cells has been predominantly attributed to TLRs; in this study, the novel role of CLRs in the BP-mast cell interaction is highlighted.

Endocrine disrupting chemicals in fish bile: a rapid method of analysis using English sole (Parophrys vetulus) from Puget Sound, WA, USA.

This study describes a recently developed and rapid method to measure bisphenol A (BPA), 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2) in bile of fish using enzymatic hydrolysis of samples followed by solid-phase extraction and ultra-performance liquid chromatography with tandem mass spectrometry. The limits of quantitation (LOQ) for BPA, EE2 and E2 were 6.3ngmL(-1), 12.5ngmL(-1) and 6.3ngmL(-1), respectively. These compounds were analyzed in bile of male English sole (Parophrys vetulus) collected from urban and non-urban sites in Puget Sound, WA, USA. The BPA and E2 concentrations (and occurrence) ranged from

Immunomodulatory molecules of Fasciola hepatica: candidates for both vaccine and immunotherapeutic development.

The liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle), a global disease that is also an important infection of humans. As soon as the parasite invades the gut wall its interaction with various host immune cells (e.g. dendritic cells, macrophages and mast cells) is complex. The parasite secretes a myriad of molecules that direct the immune response towards a favourable non-protective Th2-mediate/regulatory environment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1), are under development as the first generation of fluke vaccines. However, this peptidase and other molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treat immune-related conditions in humans and animals.

The effects of Fasciola hepatica tegumental antigens on mast cell function.

Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6(-/-) mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6(-/-) independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.