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.

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.

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.

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.

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 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.

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 tegumental coat impairs mast cells' ability to drive Th1 immune responses.

The parasitic worm Fasciola hepatica induces strong Th2 and T-regulatory immune responses while simultaneously suppressing Th1-driven immune responses to bystander microbial infections. It also prevents the initiation of Th1-mediated autoimmune disorders in mice through the suppression of Th17 and Th1 immune responses, and this can be mimicked by parasite-derived molecules. We have isolated F. hepatica tegumental coat Ag (FhTeg) and demonstrated its suppressive effect in vivo by directly targeting dendritic cells, impairing their ability to drive Th1 responses. Mast cells are critical in promoting Th1 protective immunity during bacterial infection and in driving Th1-mediated pathological conditions in autoimmune diseases. In this article, we show that FhTeg inhibits the ability of mast cells to drive the Th1 immune response by suppressing cytokine secretion (TNF-α, IL-6, IFN-γ, and IL-10) and ICAM1 expression in mast cells stimulated with LPS or heat-inactivated Bordetella pertussis Ag. These heat-inactivated B. pertussis Ag/LPS-stimulated mast cells fail to promote Th1 immune responses in CD4(+) T cells when pretreated with FhTeg, and a role for ICAM1 in this process was demonstrated. FhTeg suppresses the activation of transcription factors in the TLR signaling pathway, which explains the decrease in cytokine production and cell surface marker expression. We demonstrated that FhTeg suppresses MAPK and NF-κB activation and enhances SOCS3 expression, which could explain its negative effect on the TLR pathways. We conclude that FhTeg targets innate immune cells, inhibiting their ability to drive Th1 immune responses.

Plasma cytokines, chemokines and cellular immune responses in pre-school Nigerian children infected with Plasmodium falciparum.

BACKGROUND: Malaria is a major cause of morbidity and mortality worldwide with over one million deaths annually, particularly in children under five years. This study was the first to examine plasma cytokines, chemokines and cellular immune responses in pre-school Nigerian children infected with Plasmodium falciparum from four semi-urban villages near Ile-Ife, Osun State, Nigeria. METHODS: Blood was obtained from 231 children (aged 39-73 months) who were classified according to mean P. falciparum density per µl of blood (uninfected (n = 89), low density (<1,000, n = 51), medium density (1,000-10,000, n = 65) and high density (>10,000, n = 22)). IL-12p70, IL-10, Nitric oxide, IFN-γ, TNF, IL-17, IL-4 and TGF-ß, C-C chemokine RANTES, MMP-8 and TIMP-1 were measured in plasma. Peripheral blood mononuclear cells were obtained and examined markers of innate immune cells (CD14, CD36, CD56, CD54, CD11c AND HLA-DR). T-cell sub-populations (CD4, CD3 and γδTCR) were intracellularly stained for IL-10, IFN-γ and TNF following polyclonal stimulation or stimulated with malaria parasites. Ascaris lumbricoides was endemic in these villages and all data were analysed taking into account the potential impact of bystander helminth infection. All data were analysed using SPSS 15 for windows and in all tests, p <0.05 was deemed significant. RESULTS: The level of P. falciparum parasitaemia was positively associated with plasma IL-10 and negatively associated with IL-12p70. The percentage of monocytes was significantly decreased in malaria-infected individuals while malaria parasitaemia was positively associated with increasing percentages of CD54+, CD11c+ and CD56+ cell populations. No association was observed in cytokine expression in mitogen-activated T-cell populations between groups and no malaria specific immune responses were detected. Although A. lumbricoides is endemic in these villages, an analysis of the data showed no impact of this helminth infection on P. falciparum parasitaemia or on immune responses associated with P. falciparum infection. CONCLUSIONS: These findings indicate that Nigerian children infected with P. falciparum exhibit immune responses associated with active malaria infection and these responses were positively associated with increased P. falciparum parasitaemia.

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.

Mast cells cultured from IL-3-treated mice show impaired responses to bacterial antigen stimulation.

OBJECTIVE AND DESIGN: This study exploits the biological activity of interleukin (IL)-3 to generate high yields of peritoneal mast cells ex vivo in order to examine pro-inflammatory immune responses in ex-vivo culture. MATERIAL OR SUBJECTS: Mast cells were obtained from the peritoneal cavity of C57BL/6 mice. TREATMENT: Mice were injected intraperitoneally twice per day for 5 days with IL-3 (40-50 µg/ml) to increase mast cell numbers. METHODS: Histological studies examined mast cell numbers in the peritoneal cavity, intestine, lung, spleen and skeletal muscle. Peritoneal mast cells cultured ex vivo (PCMCs) were stimulated for 24 h with lipopolysaccharide and Bordetella pertussis antigen and secretion of tumour necrosis factor-α, IL-6, IL-4, IL-5, IL-10 and interferon-γ into supernatant was measured by commercial ELISA. Cell surface marker expression of FcεRI, c-kit, OX40L and TLR2 was measured by flow cytometry. Mast cell degranulation was measured using a ß-hexosaminidase assay. RESULTS: IL-3 treatment increases mast cell numbers in the peritoneal cavity, spleen and muscle but not intestine and lung of C57BL/6 mice. PCMCs generated from IL-3-treated mice exhibit impaired growth, differentiation and responses to activation as measured by decreased cytokine secretion and cell surface marker expression. CONCLUSION: Mast cells cultured from IL-3-treated mice show impaired responses.

Helminth cysteine proteases inhibit TRIF-dependent activation of macrophages via degradation of TLR3.

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.

Major secretory antigens of the helminth Fasciola hepatica activate a suppressive dendritic cell phenotype that attenuates Th17 cells but fails to activate Th2 immune responses.

Fasciola hepatica is a helminth pathogen that drives Th2/Treg immune responses in its mammalian host. The parasite releases a large number of molecules that are critical to inducing this type of immune response. Here we have selected recombinant forms of two major F. hepatica secreted molecules, the protease cathepsin L (rFhCL1) and an antioxidant, sigma class glutathione transferase (rFhGST-si), to examine their interactions with dendritic cells (DCs). Despite enzymatic and functional differences between these molecules, both induced interleukin-6 (IL-6), IL-12p40, and macrophage inflammatory protein 2 (MIP-2) secretion from DCs and enhanced CD40 expression. While this induction was mediated by Toll-like receptor 4 (TLR4), the subsequent intracellular signaling pathways differed; rFhCL1 signaled through p38, and rFhGST-si mediated its effect via c-Jun N-terminal kinase (JNK), p38, p-NF-kappaBp65, and IRF5. Neither rFhCL1 nor rFhGST-si enhanced DC phagocytosis or induced Th2 immune responses in vivo. However, DCs matured in the presence of either enzyme attenuated IL-17 production from OVA peptide-specific T cells in vivo. In addition, DCs exposed to either antigen secreted reduced levels of IL-23. Therefore, both F. hepatica FhCL1 and FhGST-si modulate host immunity by suppressing responses associated with chronic inflammation-an immune modulatory mechanism that may benefit the parasite's survival within the host.

Impact of repeated four-monthly anthelmintic treatment on Plasmodium infection in preschool children: a double-blind placebo-controlled randomized trial.

BACKGROUND: Helminth infections can alter susceptibility to malaria. Studies need to determine whether or not deworming programs can impact on Plasmodium infections in preschool children. METHODS: A double-blind placebo-controlled randomised trial was conducted to investigate the impact of anthelmintic treatment on Plasmodium infection in children aged 12-59 months. Children were randomly assigned to receive either albendazole or placebo every four months for 12 months with a follow-up at 14 months. RESULTS: 320 Children (out of 1228, 26.1%) complied with all the follow-up assessments. Plasmodium prevalence and mean Plasmodium parasite density was significantly higher in the treatment group (44.9% and 2319 ± SE 511) compared to the placebo group (33.3% and 1471 ± 341) at baseline. The odds of having Plasmodium infection increased over time for children in both the placebo and treatment groups, however this increase was significantly slower for children in the treatment group (P = 0.002). By month 14, mean Plasmodium density had increased by 156% in the placebo group and 98% in the treatment group but the rate of change in Plasmodium density was not significantly different between the groups. The change from baseline in haemoglobin had a steeper increase among children in the treatment group when compared to the placebo group but this was not statistically significant. CONCLUSIONS: Repeated four-monthly anthelminthic treatments for 14 months resulted in a significantly lower increase in the prevalence of Plasmodium infection in preschool children which coincided with a reduction in both the prevalence and intensity of A. lumbricoides infections. TRIAL REGISTRATION: Current controlled trials ISRCTN44215995.

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.

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.

The Fasciola hepatica tegumental antigen suppresses dendritic cell maturation and function.

Parasitic worms and molecules derived from them have powerful anti-inflammatory properties and are shown to have therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress antigen-specific Th1 responses in concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. Here, F. hepatica tegumental antigen (Teg) was shown to significantly suppress serum levels of gamma interferon (IFN-gamma) and interleukin-12p70 (IL-12p70) in a model of septic shock. Since dendritic cells (DCs) are a good source of IL-12p70 and critical in driving adaptive immunity, we investigated the effects of F. hepatica Teg on the activation and function of murine DCs. While Teg alone did not induce cytokine production or cell surface marker expression on DCs, it significantly suppressed cytokine production (IL-12p70, IL-6, IL-10, tumor necrosis factor alpha, and nitrite) and cell surface marker expression (CD80, CD86, and CD40) in DCs matured with a range of Toll-like receptor (TLR) and non-TLR ligands. Teg works independently of the TLR4 pathway, since it still functioned in DCs generated from TLR4 mutant and knockout mice. It impaired DC function by inhibiting their phagocytic capacity and their ability to prime T cells. It does not appear to target the common components (extracellular signal-regulated kinase, Jun N-terminal protein kinase, or p38) of the TLR pathways; however, it suppressed the active p65 subunit of the transcription factor NF-kappaB in mature DCs, which could explain the impairment of proinflammatory cytokine production. Overall, our results demonstrate the potent anti-inflammatory properties of F. hepatica Teg and its therapeutic potential as an anti-inflammatory agent.

Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages.

During helminth infections, alternatively activated macrophages (AAMacs) are key to promoting Th2 responses and suppressing Th1-driven inflammatory pathology. Th2 cytokines IL-4 and/or IL-13 are believed to be important in the induction and activation of AAMacs. Using murine models for the helminth infections caused by Fasciola hepatica (Fh) and Schistosoma mansoni (Sm), we show that a secreted antioxidant, peroxiredoxin (Prx), induces alternative activation of macrophages. These activated, Ym1-expressing macrophages enhanced the secretion of IL-4, IL-5, and IL-13 from naive CD4(+) T cells. Administration of recombinant FhPrx and SmPrx to wild-type and IL-4(-/-) and IL-13(-/-) mice induced the production of AAMacs. In addition, Prx stimulated the expression of markers of AAMacs (particularly, Ym1) in vitro, and therefore can act independently of IL-4/IL-13 signaling. The immunomodulatory property of Prx is not due to its antioxidant activity, as an inactive recombinant variant with active site Cys residues replaced by Gly could also induce AAMacs and Th2 responses. Immunization of mice with recombinant Prx or passive transfer of anti-Prx antibodies prior to infection with Fh not only blocked the induction of AAMacs but also the development of parasite-specific Th2 responses. We propose that Prx activates macrophages as an initial step in the induction of Th2 responses by helminth parasites and is thereby a novel pathogen-associated molecular pattern.

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.