Pseudocoelomic Fluid Research Articles

Oral administration of helminth fluid modulates distinct tuft cell and immune-metabolic cues linked to reduced body fat.

Intestinal tuft cells have been shown to induce type 2 immune responses during viable parasite infections, but whether oral supplementation with a parasitic exudate is able to promote type 2 immune responses that have been shown to positively regulate obesogenic metabolic processes is yet unresolved. High-fat fed mice were gavaged with pseudocoelomic fluid (PCF) derived from the helminth Ascaris suum or saline thrice a week during weeks 5-9, followed by examination of intestinal tuft cell activity, immune, and metabolic parameters. Helminth PCF upregulated expression of distinct genes in small intestinal tuft cells, including genes involved in regulation of RUNX1 and organic cation transporters. Helminth PCF also enhanced levels of innate lymphoid cells in the ileum, and eosinophils in epididymal white adipose tissue (eWAT). Network analyses revealed two distinct immunometabolic cues affected by oral helminth PCF in high-fat fed mice: one coupling the small intestinal tuft cell responses to the fat-to-lean mass ratio and a second coupling eosinophils in eWAT to general regulation of body fat mass. Our findings point to specific mechanisms by which oral supplementation with helminth PCF may translate into systems-wide effects linking to reduced body and fat mass gain in mice during high-fat feeding.

Bma-LAD-2, an Intestinal Cell Adhesion Protein, as a Potential Therapeutic Target for Lymphatic Filariasis.

ABSTRACTLymphatic filariasis is a debilitating disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Despite substantial success, efforts to eliminate LF will likely require more time and resources than predicted. Identifying new drug and vaccine targets in adult filariae could help elimination efforts. This study’s aim was to evaluate intestinal proteins in adult Brugia malayi worms as possible therapeutic targets. Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was determined to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1 day post-siRNA incubation and an 87.02% reduction in protein expression 2 days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6 days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult Brugia malayi and may be an effective therapeutic target.

Ascaris suum Informs Extrasynaptic Volume Transmission in Nematodes.

Neural circuit synaptic connectivities (the connectome) provide the anatomical foundation for our understanding of nematode nervous system function. However, other nonsynaptic routes of communication are known in invertebrates including extrasynaptic volume transmission (EVT), which enables short- and/or long-range communication in the absence of synaptic connections. Although EVT has been highlighted as a facet of Caenorhabditis elegans neurosignaling, no experimental evidence identifies body cavity fluid (pseudocoelomic fluid; PCF) as a vehicle for either neuropeptide or biogenic amine transmission. In the parasitic nematode Ascaris suum, FMRFamide-like peptides encoded on flp-18 potently stimulate female reproductive organs but are expressed in cells that are anatomically distant from the reproductive organ, with no known synaptic connections to this tissue. Here we investigate nonsynaptic neuropeptide signaling in nematodes mediated by the body cavity fluid. Our data show that (i) A. suum PCF (As-PCF) contains a catalog of neuropeptides including FMRFamide-like peptides and neuropeptide-like proteins, (ii) the A. suum FMRFamide-like peptide As-FLP-18A dominates the As-PCF peptidome, (iii) As-PCF potently modulates nematode reproductive muscle function ex vivo, mirroring the effects of synthetic FLP-18 peptides, (iv) As-PCF activates the C. elegans FLP-18 receptors NPR-4 and -5, (v) As-PCF alters C. elegans behavior, and (vi) FLP-18 and FLP-18 receptors display pan-phylum distribution in nematodes. This study provides the first direct experimental evidence to support an extrasynaptic volume route for neuropeptide transmission in nematodes. These data indicate nonsynaptic signaling within the nematode functional connectome and are particularly pertinent to receptor deorphanization approaches underpinning drug discovery programs for nematode pathogens.

Molecular cloning, expression and characterization of aspartyl protease inhibitor from Ancylostoma ceylanicum.

Aspartyl protease inhibitors (APIs) from parasitic intestinal nematodes are highly immunogenic and have been suggested as potential vaccine antigens. Ac-API-1 from Ancylostoma caninum showed strong immunogenicity and its polyclonal antibodies could specifically recognize the excretory/secretory products of adult worms. However, little is known about molecular characteristics and biological function of API from Ancylostoma ceylanicum (Ace-API). In this study, the Ace-API mature peptide coding sequence was cloned and expressed, and molecular characteristics of its full length sequence were analyzed. Ace-API cDNA was 684bp in length, which encoded 228 amino acids. The similarity of the Ace-API amino acid sequence to Ac-API-1 and Adu-API-1 was 96.93% and 96.49%, respectively, and they clustered together in the phylogenetic tree. Escheria coli-expressed recombinant protein was mainly soluble in the supernatant of bacterial cell lysate. Western blot showed that Ace-API protein had good reactivity to the serum of infected dogs. Pepsin inhibition assay revealed that the recombinant protein had inhibitory activity on pepsin. Immunofluorescence results demonstrated that Ace-API was mainly localized to the epidermis, excretory glands, and pseudocoelomic fluid of the adult. Using the quantitative real-time PCR, the expression of Ace-api mRNA in adults was significantly higher than that in the third stage (L3) larvae. Together, these data indicate that Ace-API is secreted extracellularly by the parasite, and might play a role in protecting the parasite against the proteolytic digestion by the host proteases, which stimulate further studies to explore this protein as a potential hookworm vaccine candidate.

Evaluation of serology to measure exposure of piglets to Ascaris suum during the nursery phase

The aim of this study was to evaluate whether serology can be used to measure exposure of piglets to Ascaris suum during the nursery phase. Experimental infection studies were performed in which 7 groups of 10 piglets of 4 weeks of age were orally infected with either 10, 20, 40, 60, 80, 100 and 500 A. suum eggs/day during 7 consecutive weeks, mimicking a nursery phase in an A. suum contaminated environment. Serum was collected on a weekly basis to monitor seroconversion on 2 ELISA tests based on the antibody recognition of either a haemoglobin protein purified from the pseudocoelomic fluid of adult A. suum or a water-soluble complete homogenate of the 3rd stage larvae isolated from the lungs (L3-lung). A dose-dependent seroconversion was measurable with the L3-lung ELISA starting from 4 weeks post-infection onwards, whereas this was not measurable with the haemoglobin-based test. After 7 weeks, equivalent to the end of the nursery phase, the L3-lung ELISA showed a 99% specificity and a 90% sensitivity to detect exposure of piglets to A. suum, with a minimum infection level of 20 A. suum eggs per day. To further evaluate the test under field conditions, a seroprevalence study was performed by sampling 10 piglets on 68 different nursery farms in Belgium. The results showed that for 38% of the farms analysed all piglets tested seronegative, whereas for the remaining 62% of the farms the percentage seropositive piglets ranged from 10 to 100%. This indicates contamination of the nursery facilities with A. suum eggs. In conclusion, the outcome of this study shows that serology can be used to measure exposure of nursery piglets to A. suum, thereby providing an additional tool in the control of this widespread parasite.

The secreted MSP domain of C. elegans VAPB homolog VPR-1 patterns the adult striated muscle mitochondrial reticulum via SMN-1

The major sperm protein domain (MSPd) has an extracellular signaling function implicated in amyotrophic lateral sclerosis. Secreted MSPds derived from the C. elegans VAPB homolog VPR-1 promote mitochondrial localization to actin-rich I-bands in body wall muscle. Here we show that the nervous system and germ line are key MSPd secretion tissues. MSPd signals are transduced through the CLR-1 Lar-like tyrosine phosphatase receptor. We show that CLR-1 is expressed throughout the muscle plasma membrane, where it is accessible to MSPd within the pseudocoelomic fluid. MSPd signaling is sufficient to remodel the muscle mitochondrial reticulum during adulthood. An RNAi suppressor screen identified survival of motor neuron 1 (SMN-1) as a downstream effector. SMN-1 acts in muscle, where it colocalizes at myofilaments with ARX-2, a component of the Arp2/3 actin-nucleation complex. Genetic studies suggest that SMN-1 promotes Arp2/3 activity important for localizing mitochondria to I-bands. Our results support the model that VAPB homologs are circulating hormones that pattern the striated muscle mitochondrial reticulum. This function is crucial in adults and requires SMN-1 in muscle, likely independent of its role in pre-mRNA splicing.

The secreted MSP domain of C. elegans VAPB homolog VPR-1 patterns the adult striated muscle mitochondrial reticulum via SMN-1.

The major sperm protein domain (MSPd) has an extracellular signaling function implicated in amyotrophic lateral sclerosis. Secreted MSPds derived from the C. elegans VAPB homolog VPR-1 promote mitochondrial localization to actin-rich I-bands in body wall muscle. Here we show that the nervous system and germ line are key MSPd secretion tissues. MSPd signals are transduced through the CLR-1 Lar-like tyrosine phosphatase receptor. We show that CLR-1 is expressed throughout the muscle plasma membrane, where it is accessible to MSPd within the pseudocoelomic fluid. MSPd signaling is sufficient to remodel the muscle mitochondrial reticulum during adulthood. An RNAi suppressor screen identified survival of motor neuron 1 (SMN-1) as a downstream effector. SMN-1 acts in muscle, where it colocalizes at myofilaments with ARX-2, a component of the Arp2/3 actin-nucleation complex. Genetic studies suggest that SMN-1 promotes Arp2/3 activity important for localizing mitochondria to I-bands. Our results support the model that VAPB homologs are circulating hormones that pattern the striated muscle mitochondrial reticulum. This function is crucial in adults and requires SMN-1 in muscle, likely independent of its role in pre-mRNA splicing.

ANISERP: a new serpin from the parasite Anisakis simplex.

BackgroundSerine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP).MethodsThe full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted.ResultsThe AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity.ConclusionsOur findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.

Ionic regulation in the Antarctic nematode Panagrolaimus davidi, measured using electron probe X-ray microanalysis

The element composition of the pseudocoelomic fluid of the Antarctic nematode Panagrolaimus davidi was analysed by electron probe X-ray microanalysis after absorbing the fluid into Sephadex G-25 beads, and after producing calibration curves by analysing various concentrations of elements of interest absorbed into beads. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. When external concentrations of specific ions were elevated there was evidence for the regulation of internal concentrations of sodium, potassium, magnesium and chlorine. The time course of changes in response to exposure to elevated levels of KCl shows an increase in internal concentrations of potassium and chlorine up to 2h after exposure, followed by a decline. This is consistent with a model of ionic regulation proposed for Caenorhabditis elegans which suggests that high concentrations of ionic osmolytes are replaced by compatible organic osmolytes.

Vampiric Isolation of Extracellular Fluid from <em>Caenorhabditis elegans</em>

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from <em>Caenorhabditis elegans</em>

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Osmoregulation in the Antarctic nematodePanagrolaimus davidi

A technique for determining the internal osmotic concentration of a small nematode using a modified nanolitre osmometer is described and used to investigate osmoregulation in the Antarctic nematode Panagrolaimus davidi. This technique enables the osmotic concentration to the measured with an accuracy of +/-12 mmol kg(-1). The pattern of melting in the nematode's different body compartments suggests that it is the osmolality of its pseudocoelomic fluid that is being measured. Panagrolaimus davidi maintains its internal osmotic concentration above that of the external medium and is thus an hyperosmotic regulator. The nematode achieves regulation under hyposmotic stress more rapidly than under hyperosmotic stress.

Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi – Comparison with other strongylid nematodes and Caenorhabditis elegans

The transthyretin-like ( ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi ( Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes ( Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory–secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic–bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing β-sheets, arranged in a β-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.

The parasitic nematode Ascaris suum secretes a hemoglobin that scavenges host derived free radicals and activates dendritic cells through TLR4

Ascaris lumbricoides is the most successful geohelminth since it currently infects >1.5 billion people world‐wide. Here,we characterize a readily available source of soluble products in the pseudo‐coelomic fluid (PCF) from adult A. suum. A 42 kDa Ascaris suum hemoglobin (AsHb)was recognized by sera from infected pigs and was identified in the muscle and intestinal tissues and the excretory/secretory products obtained from third and fourth‐stage larvae (L3/L4) and adult worms. Comparative sequence information indicated sequence homology in parasitic but not free living nematodes. Exposure of BMDC to AsHb induced the maturation markers CD40, CD86, and MHCII. Co‐stimulation of BMDC with AsHb and LPS decreased IL‐12 production and significantly increased IL‐10 compared to stimulation with LPS alone. AsHb‐dependent maturation and IL‐10 production were abrogated in BMDC from TLR4‐deficient mice. In addition,confocal studies demonstrate co‐localization of AsHb with TLR4 in mouse BMDC, followed by internalization of AsHb through a receptor‐mediated endocytic pathway. Finally, AsHb was able to remove extracellular free radical reductants produced by activated swine bronchial‐alveolar cells.

C. elegansDisabled is required for cell-type specific endocytosis and is essential in animals lacking the AP-3 adaptor complex

Disabled proteins are a conserved family of monomeric adaptor proteins that in mammals are implicated in the endocytosis of lipoprotein receptors. Previous studies have shown that the sole Caenorhabditis elegans Disabled homologue, DAB-1, is involved in the lipoprotein receptor-mediated secretion of a fibroblast growth factor. We show here that DAB-1 is essential for the uptake of yolk protein by developing oocytes, and for the localisation of the yolk receptor RME-2. The localisation of DAB-1 in oocytes is itself dependent upon clathrin and AP2, consistent with DAB-1 acting as a clathrin-associated sorting protein during yolk protein endocytosis. DAB-1 is also required for the endocytosis of molecules from the pseudocoelomic fluid by the macrophage-like coelomocytes, and is broadly expressed in epithelial tissues, consistent with a general role in receptor-mediated endocytosis. We also show that dab-1 mutations are synthetic lethal in combination with loss-of-function mutations affecting the AP-1 and AP-3 complexes, suggesting that the reduced fluid and membrane uptake exhibited by dab-1 mutants sensitises them to defects in other trafficking pathways.

The Immunomodulatory Effects of Ascaris suum Pseudocoelomic Fluid in Th1-Mediated Systems (90.10)

Abstract Recent studies found that adoptive transfer of helminth-primed dendritic cells impairs host protection to the enteric pathogen, Citrobacter rodentium. In the present study, we used Ascaris suum pseudocoelomic fluid (PCF) in lieu of infection, and demonstrated that pre-exposure of bone-marrow derived dendritic cells to PCF, prior to LPS stimulation, suppressed activation marker expression and production of IL-12. Similar to LPS, heat-inactivated and extract preparations of C. rodentium induced dendritic cell activation. This activation was suppressed in cells previously exposed to PCF, resulting in decreased expression of CD40, CD86, and MHC II. These findings suggest that PCF may modify Th1 responses in the host. In preliminary studies, BALB/c mice were sensitized with PCF (100 μg), followed by oral inoculation with C. rodentium (2.5 x 108 CFU). Bacterial colonization and colonic hyperplasia was assessed at days 3 and 7 post-infection. Mice receiving two sensitizations prior to inoculation had increased C. rodentium colonization in the cecum and colon by day 7 post-infection, compared to those receiving no pre-treatment. We are currently repeating this experiment using a higher concentration of PCF (200 μg) to enhance differences in bacterial colonization. This model may help determine if PCF alters the course of Th1-mediated immunoregulation in allergic inflammatory responses.

Toll-like Receptor 4-Dependent activation of Dendritic cells by heme binding protein from Ascaris suum (90.9)

Abstract Toll-like receptors (TLRs) recognize specific molecular patterns on invading pathogens leading to the development of host immunity. A number of pathogens including helminths have used pattern recognition by TLRs to modulate host immunity and inflammation to establish a chronic infection. To understand the molecular basis for the TLR-induced tolerance induction observed in individuals infected with helminth parasites, we have established a mouse model combining allergic inflammation and Ascaris suum infection. These in vivo studies demonstrated that concomitant infection with chronic A. suum can inhibit allergic inflammation in response to a non-parasite allergen, ragweed (RW). In the current study, we have purified heme-binding protein (HBP) from pseudocoelomic fluid of A.suum and studied its antigenic and immunomodulatory properties using bone marrow derived dendritic cells. Recognition of HBP by antiserum from pigs infected with Ascaris larvae indicate that HBP is one of the antigenic proteins present in PCF. Furthermore, HBP exposure increases activation marker surface expression on DC including CD40, CD86 and MHC II. This increase in activation marker expression is abrogated in TLR4, but not TLR2, knockout mice implicating that HBP is a TLR4 ligand. Pretreatment of dendritic cells with HBP prior to LPS stimulation results in increased production of IL-10 but not IL-12 compared with DC exposed to LPS alone. These results provide the first evidence for a novel TLR4-mediated response to Ascaris suum HBP resulting in altered APC activation and cytokine production.

Ascaris Antigens Suppress Experimental Allergic Inflammation

RATIONALE: Evaluate the potential of pseudocoelomic fluid (PCF) from Ascaris in modulating immune responses to ragweed allergen using a mouse model.METHODS: In order to characterize the allergic immunosuppression by A. suum antigens, we have adopted a two-pronged in vivo and in vitro approach. We assessed the in vivo effects of PCF and ABA-1, the predominant protein in PCF, in a ragweed sensitized mouse model of asthma using histological examination. IL-4, IL-5 and IL-13 cytokine levels assessed using multiplex bead immunoassay in BAL fluid. In vitro studies using bone marrow-derived dendritic cells (bmDC) were employed to evaluate the suppression of co-stimulatory molecules such as CD40 and CD86 in response to PCF by flow cytometry. Production of Th1/Th2 cytokines by bmDCs in response to ABA-1 and PCF exposure was assessed using multiplex bead immunoassay.RESULTS: Protection could be seen by significantly decreased cell numbers in BALF, decreased eosinophilic infiltration and reduced lung pathology in RW/PCF and RW/ABA-1 group compared to RW alone group. IL-5 and IL-13 levels in BALF were significantly reduced in PCF group compared to RW alone. PCF was able to down-regulate the expression of costimulatory molecules and production of IL-12 by dendritic cells in response to LPS in vitro demonstrating its immunosuppressive properties. Inactivation of proteins lead to increased bmDC activation as evidenced by upregulation of CD40 and CD86 as well as increased IL-12 production. These findings suggested that the protein component of PCF might play a role in protection.CONCLUSIONS:A. suum antigens appear to modulate allergic inflammation and potential regulatory mechanisms are currently under investigation. RATIONALE: Evaluate the potential of pseudocoelomic fluid (PCF) from Ascaris in modulating immune responses to ragweed allergen using a mouse model. METHODS: In order to characterize the allergic immunosuppression by A. suum antigens, we have adopted a two-pronged in vivo and in vitro approach. We assessed the in vivo effects of PCF and ABA-1, the predominant protein in PCF, in a ragweed sensitized mouse model of asthma using histological examination. IL-4, IL-5 and IL-13 cytokine levels assessed using multiplex bead immunoassay in BAL fluid. In vitro studies using bone marrow-derived dendritic cells (bmDC) were employed to evaluate the suppression of co-stimulatory molecules such as CD40 and CD86 in response to PCF by flow cytometry. Production of Th1/Th2 cytokines by bmDCs in response to ABA-1 and PCF exposure was assessed using multiplex bead immunoassay. RESULTS: Protection could be seen by significantly decreased cell numbers in BALF, decreased eosinophilic infiltration and reduced lung pathology in RW/PCF and RW/ABA-1 group compared to RW alone group. IL-5 and IL-13 levels in BALF were significantly reduced in PCF group compared to RW alone. PCF was able to down-regulate the expression of costimulatory molecules and production of IL-12 by dendritic cells in response to LPS in vitro demonstrating its immunosuppressive properties. Inactivation of proteins lead to increased bmDC activation as evidenced by upregulation of CD40 and CD86 as well as increased IL-12 production. These findings suggested that the protein component of PCF might play a role in protection. CONCLUSIONS:A. suum antigens appear to modulate allergic inflammation and potential regulatory mechanisms are currently under investigation.

Fluoxetine-Resistance Genes inCaenorhabditis elegansFunction in the Intestine and May Act in Drug Transport

Fluoxetine (Prozac) is one of the most widely prescribed pharmaceuticals, yet important aspects of its mechanism of action remain unknown. We previously reported that fluoxetine and related antidepressants induce nose muscle contraction of C. elegans. We also reported the identification and initial characterization of mutations in seven C. elegans genes that cause defects in this response (Nrf, nose resistant to fluoxetine). Here we present genetic evidence that the known nrf genes can be divided into two subgroups that confer sensitivity to fluoxetine-induced nose contraction by distinct pathways. Using both tissue-specific promoters and genetic mosaic analysis, we show that a gene from one of these classes, nrf-6, functions in the intestine to confer fluoxetine sensitivity. Finally, we molecularly identify nrf-5, another gene in the same class. The NRF-5 protein is homologous to a family of secreted lipid-binding proteins with broad ligand specificity. NRF-5 is expressed in the intestine and is likely secreted into the pseudocoelomic fluid, where it could function to transport fluoxetine. One model that explains these findings is that NRF-5 binds fluoxetine and influences its presentation or availability to in vivo targets.

Cloning and characterisation of an aspartyl protease inhibitor (API-1) from Ancylostoma hookworms

Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5′ and 3′ ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878 bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme–inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine.