Identification of cross-reactive markers to strengthen the development of immunodiagnostic methods for angiostrongyliasis and other parasitic infections.

Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.

Physiology and immunity of the invasive giant African snail, Achatina (Lissachatina) fulica, intermediate host of Angiostrongylus cantonensis.

As one of the most successful invasive land snail species, Achatina (Lissachatina) fulica Bowdich, 1822 has achieved wide global distribution, particularly in (sub)tropical regions, with further dispersal likely due to climate change. This species of giant African snails (up to 17 cm shell length) is a pest that has extensive negative impact on agriculture and can serve as vector for several parasites, including Angiostrongylus cantonensis, a nematode parasite that causes (human) eosinophilic meningitis, an emergent disease. Investigation showed that A. cantonensis infection negatively impacts the metabolism of A. fulica by depleting polysaccharide stores of the intermediate host, compromising the energy balance of the snail. A review of the literature indicates that A. fulica possesses potent innate type immune defenses to counter infection, including phagocytic hemocytes capable of deploying reactive oxygen species and lectins for non-self recognition, a serine protease-dependent coagulation response (not observed in other taxa of gastropods), as well as antimicrobial proteins including achacin, an antimicrobial protein. A recent chromosome level genome assembly will facilitate progressively detailed characterization of these immune features of A. fulica. We strongly encourage further immunological studies of A. fulica, ranging from organismal level to molecular biology to gain better understanding of the A. fulica internal defense response to nematode pathogens like A. cantonensis and the contribution of immune function to the invasiveness of (snail) species. Characterization of immunity of A. fulica, representing the understudied Stylommatophora (panpulmonate landsnails) will also broaden the comparative immunology of Gastropoda.

Nippostrongylus brasiliensis infection inhibits hippocampal neurogenesis in mice.

The brain has long been considered a site of "immune privilege"; however, recent evidence indicates the presence of brain-immune interactions in physiological and pathological conditions. Neurogenesis, a process of generating functionally integrated neurons, occurs in the adult brain of mammals. The adult neurogenesis predominantly takes place in the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ). Several studies have shown that an immune reaction or alteration could affect adult neurogenesis activity, suggesting a link between the immune system and adult neurogenesis. Helminth infection is one of the activators of Th2 immune response. However, the influence of this type of immune reaction on adult neurogenesis is not well studied. In this study, we evaluated adult neurogenesis in mice infected with the helminth Nippostrongylus brasiliensis (Nb). Immunohistochemically, the number of both doublecortin-positive cells and doublecortin/5-bromodeoxyuridine (BrdU)-double-positive cells was decreased in the SGZ of Nb-infected mice by day 9 after infection. However, the total number of BrdU-positive newborn cells in the SGZ did not change. In no significant alterations were detected in the SVZ of infected mice. In addition, using reverse transcription-quantitative polymerase chain reaction, we observed no significant changes in the expression levels of neurotropic factors important for neurogenesis in the hippocampus. In conclusion, our results indicate that adult neurogenesis in SGZ, but not in SVZ, is inhibited by Nb infection. Th2 immune response might have a suppressive effect on hippocampal neurogenesis.

Nippostrongylus brasiliensis infection leads to impaired reference memory and myeloid cell interference.

Hookworm infection is endemic in developing countries, leading to poor cognitive function-among other disruptions. In this study, the effects of Nippostrongylus brasiliensis infection (a murine model of Necator Americanus) on cognitive function were investigated. Though impaired cognition has been extensively reported, the exact domain of cognition affected is still unknown, hence requiring investigation. The objective of this study was to identify possible cognitive changes during Nippostrongylus brasiliensis infection in mice, using the Morris water maze. Here, we show for the first time that mice infected with Nippostrongylus brasiliensis were able to learn the Morris water maze task, but demonstrated impaired reference memory. Anxiety measured by thigmotaxis in the maze, did not play a role for the observed cognitive impairment. Of further interest, an increase in the number of hippocampal macrophages and microglia with training and/or infection suggested a significant role of these cell types during spatial learning. Together, these experimental mouse studies suggest that helminth infections do have an impact on cognition. Further experimental animal studies on cognition and infection might open new approaches for a better understanding and impact of pathogen infections.

The Study of Host Immune Responses Elicited by the Model Murine Hookworms Nippostrongylus brasiliensis and Heligmosomoides polygyrus.

Hookworm infections (Necator americanus or Ancylostoma duodenale) represent a major neglected tropical disease, affecting approximately 700 million people worldwide, and can cause severe morbidity due to the need for these worms to feed on host blood. N. brasiliensis and H. polygrus, both rodent parasites, are the two most commonly employed laboratory models of experimental hookworm infection. Both parasites evoke type 2 immune responses, and their use has been instrumental in generating fundamental insight into the molecular mechanisms of type-2 immunity and for understanding how the immune response can control parasite numbers. Here we provide a complete set of methods by which to investigate the natural progression of infection and the host immunological responses in the lung and intestine of H. polygyrus- and N. brasiliensis-infected mice. Detailed information is included about the most important parasitological and immunological measurements to perform at each time point. © 2017 by John Wiley & Sons, Inc.

Interleukin-13/interleukin-4 receptor pathway is crucial for production of Sda-sialomucin in mouse small intestinal mucosa by Nippostrongylus brasiliensis infection.

Mucin is a major component of mucus in gastrointestinal mucosa. Increase of specific sialomucins having Sda blood group antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, is considered to be associated with expulsion of the parasitic intestinal nematode Nippostrongylus brasiliensis. In this study, we examined the relationship between interleukin (IL)-13 pathway and expression of Sda-sialomucins in small intestinal mucosa with N. brasiliensis infection. Nematode infection induced marked increases in small intestinal mucins that reacted with anti-Sda antibody in wild type (wt) mice. However, this increase due to infection was supressed in IL-4 receptor α deficient (IL-4Rα-/-) mice, which lack both IL-4 and IL-13 signaling via IL-4R, and severe combined immunodeficient (SCID) mice, which have defects in B- and T-lymphocytes. Analysis using tandem mass spectroscopy showed that Sda-glycans were not expressed in small intestinal mucins in IL-4Rα-/- and SCID mice after infection despite the appearance of Sda-glycans in the infected wt mice. Inoculation of recombinant IL-13 into the infected SCID mice restored expression of Sda-glycan. Our results suggest that the IL-13/IL-4R axis is important for the production of Sda-sialomucins in the host intestinal mucosa with parasitic nematode infection.

Cross-reactivity of the 31 kDa antigen of Angiostrongylus cantonensis - Dealing with the immunodiagnosis of meningoencephalitis.

The primary causative agent of eosinophilic meningoencephalitis (EoM) in endemic regions is the nematode Angiostrongylus cantonensis. The occurrence of EoM was previously restricted to countries in Southeast Asia and the Pacific Islands; however, more recently, it has been reported from other regions, including Brazil. The commonly used diagnosis is detection of specific antibody reactivity to the 31 kDa antigen, which is derived from female worm somatic extracts. Here we report the occurrence of cross-reactivity to this antigen in sera from other parasitic infections, especially those that may cause EoM, such as gnathostomiasis, toxocariasis, hydatidosis and strongyloidiasis. We also demonstrated that the cross-reactivity, in part, is dependent of the concentration of antigen used in Western blot assays. We discuss the importance of these findings on the interpretation of this test.

Macrophage migration inhibitory factor deficiency enhances immune response to Nippostrongylus brasiliensis.

Infections with helminth parasites are endemic in the developing world and are a target for intervention with new therapies. Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic effects in inflammation and immune responses. We investigated the role of MIF in a naturally cleared model of helminth infection in rodents, Nippostrongylus brasiliensis. At day 7 postinfection, MIF-deficient (MIF-/-) mice had reduced parasite burden and mounted an enhanced type 2 immune response (Th2), including increased Gata3 expression and interleukin-13 (IL-13) production in the mesenteric lymph nodes (MLNs). Bone marrow reconstitution demonstrated that MIF produced from hematopoietic cells was crucial and Rag1-/- reconstitution provided direct evidence that MIF-/- CD4+ T cells were responsible for the augmented parasite clearance. MIF-/- CD4+ T cells produced less IL-6 postinfection, which correlated with enhanced Th2 responses. MIF-/- CD4+ T cells exhibited lower nuclear factor-κB activation, potentially explaining the reduction in IL-6. Finally, we demonstrated enhanced clearance of the parasite and Th2 response in wild-type mice treated with the MIF tautomerase inhibitor, sulforaphane, a compound found naturally found in cruciferous vegetables. These results are the first to describe the importance of the tautomerase enzyme activity in MIF function in N. brasiliensis infection.

Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of Nippostrongylus brasiliensis through induction of TH2 cytokines.

Despite increased appreciation for the role of nicotinic receptors in the modulation of and response to inflammation, the contribution of muscarinic receptors to mucosal homeostasis, clearance of enteric pathogens, and modulation of immune cell function remains relatively undefined. Uninfected and Nippostrongylus brasiliensis-infected wild-type and type 3 muscarinic receptor (M3R)-deficient (Chrm3(-/-)) mice were studied to determine the contribution of M3R to mucosal homeostasis as well as host defense against the TH2-eliciting enteric nematode N. brasiliensis Intestinal permeability and expression of TH1/TH17 cytokines were increased in uninfected Chrm3(-/-) small intestine. Notably, in Chrm3(-/-) mice infected with N. brasiliensis, small intestinal upregulation of TH2 cytokines was attenuated and nematode clearance was delayed. In Chrm3(-/-) mice, TH2-dependent changes in small intestinal function including smooth muscle hypercontractility, increased epithelial permeability, decreased epithelial secretion and absorption, and goblet cell expansion were absent despite N. brasiliensis infection. These findings identify an important role for M3R in host defense and clearance of N. brasiliensis, and support the expanding role of cholinergic muscarinic receptors in maintaining mucosal homeostasis.

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis.

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp(fl/fl)Cre(WT) mice that express selenoproteins driven by tRNA(Sec) (Trsp), whereas N. brasiliensis-infected Trsp(fl/fl)Cre(LysM) selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ(12)-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses.

Increased exposure to Plasmodium chabaudi antigens sustains cross-reactivity and avidity of antibodies binding Nippostrongylus brasiliensis: dissecting cross-phylum cross-reactivity in a rodent model.

Mounting an antibody response capable of discriminating amongst and appropriately targeting different parasites is crucial in host defence. However, cross-reactive antibodies that recognize (bind to) multiple parasite species are well documented. We aimed to determine if a higher inoculating dose of one species, and thus exposure to larger amounts of antigen over a longer period of time, would fine-tune responses to that species and reduce cross-reactivity. Using the Plasmodium chabaudi chabaudi (Pcc)-Nippostrongylus brasiliensis (Nb) co-infection model in BALB/c mice, in which we previously documented cross-reactive antibodies, we manipulated the inoculating dose of Pcc across 4 orders of magnitude. We investigated antigen-specific and cross-reactive antibody responses against crude and defined recombinant antigens by enzyme linked immunosorbent assay, Western blot and antibody depletion assays. Contrary to our hypothesis that increasing exposure to Pcc would reduce cross-reactivity to Nb, we found evidence for increased avidity of a subpopulation of antibodies that recognized shared antigens. Western blot indicated proteins of apparent monomer molecular mass 28 and 98 kDa in both Nb and Pcc antigen preparations and also an Nb protein of similar size to recombinant Pcc antigen, merozoite surface protein-1(19). The implications of antibodies binding antigen from such phylogenetically distinct parasites are discussed.

Type 2 immunity-dependent reduction of segmented filamentous bacteria in mice infected with the helminthic parasite Nippostrongylus brasiliensis.

BACKGROUND: Dynamic interactions between the host and gastrointestinal microbiota play an important role for local and systemic immune homeostasis. Helminthic parasites modulate the host immune response, resulting in protection against autoimmune disease but also increased susceptibility to pathogen infection. The underlying mechanisms remain largely unknown. RESULTS: We showed that the type 2 immune response to enteric Nippostrongylus brasiliensis infection in mice was associated with altered intestinal mucin and AMP expression and shifts in microbiota composition. Most strikingly, infection reduced concentrations of intestinal segmented filamentous bacteria (SFB), known inducers of T helper 17 cells, and IL-17-associated gene expression. Infected mice deficient in IL-13 or STAT6 did not reduce SFB or IL-17, and exogenous IL-25 replicated the effects of parasite infection in wild type mice. CONCLUSIONS: Our data show that parasite infection acts through host type 2 immunity to reduce intestinal SFB and expression of IL-17, providing an example of a microbiota-dependent immune modulation by parasites.

Natural and vaccine-mediated immunity to Salmonella Typhimurium is impaired by the helminth Nippostrongylus brasiliensis.

BACKGROUND: The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. METHODOLOGY/PRINCIPAL FINDINGS: Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. CONCLUSION: These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

Inducible deletion of CD28 prior to secondary nippostrongylus brasiliensis infection impairs worm expulsion and recall of protective memory CD4⁺ T cell responses.

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28⁻/⁻ mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28(-/lox)Cre⁺/⁻+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28⁻/⁻ mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28(-/lox)Cre⁺/⁻ mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28⁻/⁻ mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T(FH) cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28⁻/⁻ mice and tamoxifen treated CD28(-/lox)Cre⁺/⁻ mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28(-/lox)Cre⁺/⁻ mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.

Lung-resident CD4⁺ T cells are sufficient for IL-4Rα-dependent recall immunity to Nippostrongylus brasiliensis infection.

Immunity to Nippostrongylus brasiliensis reinfection requires pulmonary CD4⁺ T-cell responses. We examined whether secondary lymphoid recruited or pre-existing lung CD4⁺ T-cell populations coordinated this immunity. To do this, we blocked T-cell egress from lymph nodes using Fingolimod (FTY720). This impaired host ability to resolve a primary infection but did not change effectiveness of recall immunity. Associated with this effective recall immunity was the expansion and T helper type 2 polarization of a pre-existing pulmonary CD4⁺ T-cell population. LTßR-Ig (lymphotoxin beta-receptor fusion protein)-mediated disruption of stromal cell organization of immune cells did not disrupt this recall immunity, suggesting that protection was mediated by a pulmonary interstitial residing CD4⁺ T-cell population. Adoptive transfer of N. brasiliensis-experienced pulmonary CD4⁺ T cells from FTY720-treated wild-type or T-cell interleukin (IL)-4Rα-deficient mice demonstrated protection to be IL-4Rα dependent. These results show that pre-existing CD4⁺ T cells can drive effective recall immunity to N. brasiliensis infection independently of T-cell recruitment from secondary lymphoid organs.

IL-4Rα-associated antigen processing by B cells promotes immunity in Nippostrongylus brasiliensis infection.

In this study, B cell function in protective T(H)2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα⁻/⁻ mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4⁺ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88⁻/⁻ B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4⁺ T cell-mediated protective immunity against N. brasiliensis infection.

A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

BACKGROUND: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. CONCLUSION: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

High throughput sequencing of the Angiostrongylus cantonensis genome: a parasite spreading worldwide.

Angiostrongylus cantonensis is a parasitic nematode of rodents and a leading aetiological agent of eosinophilic meningitis in humans. Definitive diagnosis is difficult, often relying on immunodiagnostic methods which utilize crude antigens. New immunodiagnostic methods based on recombinant proteins are being developed, and ideally these methods would be made available worldwide. Identification of diagnostic targets, as well as studies on the biology of the parasite, are limited by a lack of molecular information on Angiostrongylus spp. available in databases. In this study we present data collected from DNA random high-throughput sequencing together with proteomic analyses and a cDNA walking methodology to identify and obtain the nucleotide or amino acid sequences of unknown immunoreactive proteins. 28 080 putative ORFs were obtained, of which 3371 had homology to other deposited protein sequences. Using the A. cantonensis genomic sequences, 156 putative ORFs, matching peptide sequences obtained from previous proteomic studies, were considered novel, with no homology to existing sequences. Full-length coding sequences of eight antigenic target proteins were obtained. In this study we generated not only the complete nucleotide sequences of the antigenic protein targets but also a large amount of genomic data which may help facilitate future genomic, proteomic, transcriptomic or metabolomic studies on Angiostrongylus.

Mucosal trapping and degradation of Nippostrongylus brasiliensis occurs in the absence of STAT6.

Hookworms represent a major infectious burden globally, especially in developing countries. The murine hookworm Nippostrongylus brasiliensis is normally cleared in a manner dependent on IL-13, IL4-R and STAT6 signalling. Here we have used STAT6-deficient animals to model a non-resistant population and describe 2 novel STAT6-independent processes for the clearance of N. brasiliensis. During primary infection STAT6-/- animals are able to clear gut-dwelling N. brasiliensis by a mechanism involving the trapping and degradation of worms in the gut mucosa. Here, a previously undescribed STAT6-independent up-regulation of Relm-ß was observed which correlated with the mucosal trapping and degradation of worms. Previous studies have indicated that during secondary infection STAT6 deficient animals fail to expel adult worms and remain susceptible to re-infection and long-term colonization of the gut. We report here that an initial partially protective response occurs early upon re-infection in the absence of STAT6, and that a late-phase protective secondary response arises in the gut of STAT6-deficient mice leading to the clearance of the majority of N. brasiliensis, through their trapping and death in the mucosal layer of the lower region of the small intestine. These findings show that there are a number of redundant effector pathways which act to reduce worm burden in the gut which can be activated by mechanisms that do not work through the dominant STAT6 signalling pathway and may be useful as targets for future vaccination strategies against resistant hookworm strains.

Intrathecal activation as a typical immune response within the central nervous system in angiostrongyliasis.

Angiostrongylus cantonensis is a zoonotic pathogen that occasionally causes human angiostrongyliasis; its main clinical manifestation is eosinophilic meningitis. This report defines the concept of intrathecal activation of complement as evidence of intrathecal synthesis of major immunoglobulins during this disease. Details are presented of the activation of complement system components in cerebrospinal fluid, and their application to our understanding of this tropical disease, which is emerging in the Western hemisphere. Intrathecal synthesis of at least one of the major immunoglobulins and a wide spectrum of patterns may be observed. Although intrathecal synthesis of C3c is always present, C4 intrathecal synthesis does not occur in every patient. The diversity of intrathecal synthesis and activation of the different complement pathways enables their division into three variant groups (A, B, and C). Variant group A includes the classical and/or lectin pathway and involves two or more major immunoglobulins with C3 and C4 intrathecal synthesis. Variant group B involves C4 in cerebrospinal fluid that comes from blood in the intrathecal activation of the classical pathway. Variant group C includes the alternative pathway.