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.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

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.

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

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.

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

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.

The screening and prioritization of contaminants of emerging concern in the marine environment based on multiple biological response measures.

There is ample evidence that a range of anthropogenic chemicals occur in the aquatic environment, some of which have the potential to cause harm. Contaminants of Emerging Concern (CECs) are a subset of anthropogenic compounds that are poorly characterized in terms of effects and occurrences, and that are generally unregulated. Due to the sheer number of chemicals used, it is necessary to identify and prioritize those that may cause biological impacts. A key challenge of doing so is the lack of traditional ecotoxicological information. The utilization of in vitro exposure-response studies or benchmarks based on in vivo data can provide a basis for developing threshold values for evaluation of potential impacts. There are challenges, including understanding the accuracy and range of application for modeled measures and translating in vitro response information from receptor models to apical endpoints. Despite this, the use of multiple lines of evidence increases the range of available information, and supports a weight-of-evidence approach to inform the screening and prioritization of CECs in the environment. The objective of this work is to perform an evaluation of CECs detected in an urban estuary, and to identify those that are most likely to elicit a biological response. Monitoring data from marine water, wastewater, and fish and shellfish tissue samples from 17 different campaigns combined with multiple biological response measures were compared with appropriate threshold values. CECs were categorized based on their potential to elicit a biological response; the uncertainty, based on consistency of lines of evidence, was also evaluated. Two-hundred-fifteen CECs were detected. Fifty-seven were identified as High Priority (likely to cause a biological effect), and 84 as Watch List (potential to cause biological effects). Due to the extent of the monitoring and range of the lines of evidence, this approach and results are applicable to other urbanized estuarine systems.

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.

A methodology for evaluating and ranking water quantity indicators in support of ecosystem-based management.

Ecosystem-based Management (EBM) is an approach that includes different management priorities and requires a balance between anthropogenic and ecological resource demands. Indicators can be used to monitor ecosystem status and trends, and assess whether projects and/or programs are leading to the achievement of management goals. As such, the careful selection of a suite of indicators is a crucial exercise. In this paper we describe an indicator evaluation and selection process designed to support the EBM approach in Puget Sound. The first step in this process was the development of a general framework for selecting indicators. The framework, designed to transparently include both scientific and policy considerations into the selection and evaluation process, was developed and then utilized in the organization and determination of a preliminary set of indicators. Next, the indicators were assessed against a set of nineteen distinct criteria that describe the model characteristics of an indicator. A literature review was performed for each indicator to determine the extent to which it satisfied each of the evaluation criteria. The result of each literature review was summarized in a numerical matrix, allowing comparison, and demonstrating the extent of scientific reliability. Finally, an approach for ranking indicators was developed to explore the effects of intended purpose on indicator selection. We identified several sets of scientifically valid and policy-relevant indicators that included metrics such as annual-7 day low flow and water system reliability, which are supportive of the EBM approach in the Puget Sound.

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.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

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.