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.

Galectin-9-like from Angiostrongylus cantonensis young adult worms modulates eosinophil chemotaxis in vitro.

BACKGROUND/PURPOSE: Eosinophils are recruited to the brain of mice after infection with Angiostrongylus canonensis. Several factors produced by infected mice are well known playing the role to chemoattract eosinophils from the blood into the brain. The purpose of this study is to investigate whether Angiostronylus cantonensis young-adult worms (AcYA) have components which have eosinophilic chemotactic activity. METHODS: Eosinophil chemotactic activity of AcYA was tested by Boyden blind-well chamber technique. The components of AcYA were analysed by SDS-PAGE and Mass spectrometry. Furthermore, galectin-9 in the cerebrospinal fluid (CSF) of infected mice and galectin-9-like in AcYA were measured by ELISA technic and also were recognized by western blot analysis respectively. RESULTS: Excretory-secretory products of AcYA did not show eosinophil chemotactic activity. However, the extracts of AcYA showed protein concentration-dependent eosinophil chemotactic activity and reached the peak at the 24 µg/ml. The eosinophil chemotactic activity was significantly reduced by lactose. The components of AcYA at molecular weights of approximatively 15 kDa and 35 kDa showed several galectins component in Mass spectrometric analysis. Furthermore, galectin-9-like in AcYA was recognized by ELISA and western blot analysis. In parallel with increase of galectin-9 in the CSF, eosinophils were also significantly increased in mouse after infected with A. cantonensis. CONCLUSION: Galectin-9-like in AcYA and galectin-9 in mouse CSF were confirmed demonstrating eosinophil chemotactic activity both in vitro study and in the infection of mouse in this study.

The opposite roles of PAS-5 and Galectin-1 in immune response during the early infection of Angiostrongylus cantonensis.

BACKGROUND: Angiostrongylus cantonensis is a human zoonotic nematode parasite. Our previous studies found that PAS-5 and Galectin-1 (Gal-1) proteins of A. cantonensis could be strongly recognized by sera from mice infected with A. cantonensis. In this study, we further evaluated the potential roles of these two proteins in the induction of immune response in mice. METHODS: Mice were immunized with recombinant PAS-5 or Gal-1 and then challenged with 30 infective A. cantonensis larvae following the last immunization. We then examined the infected mice for changes in serum antibodies and cytokines by ELISA, CD4+ T cells and CD4+CD25+FoxP3+ regulatory T cells (Tregs) by flow cytometry, and tissue damage severity by hematoxylin-eosin (H&E) staining. RESULTS: Compared with control mice, the PAS-5-immunized mice exhibited increased levels of serum antibodies and cytokines (except for IL-10) at different time points post-infection. PAS-5 immunization promoted significant proliferation of CD4+ T cells, and caused more damage in the brain tissue. Vaccination with Gal-1 inhibited the production of antibodies (except for IgG1) and IFN-γ, but promoted the expression of IL-4 and IL-10. Gal-1 immunization results in significant increases in the levels of CD4+CD25+FoxP3+ Tregs, and mild inflammatory changes. CONCLUSIONS: Taken together, our findings show that PAS-5 enhances, but Gal-1 inhibits the immune response in the early stage of A. cantonensis infections.

[Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum].

INTRODUCTION: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. OBJECTIVE: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. MATERIALS AND METHODS: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. RESULTS: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). CONCLUSIONS: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Estandarización de una prueba múltiple de reacción en cadena de la polimerasa en tiempo real para la identificación de Angiostrongylus cantonensis, A. costaricensis y A. vasorum / Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Resumen Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específica para el manejo de las angiostrongilosis humana y la animal. Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus. Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®. Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano. Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %). Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

Abstract Introduction: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. Objective: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. Materials and methods: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. Results: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). Conclusions: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Stimulation of IL-1ß and IL-6 through NF-κB and sonic hedgehog-dependent pathways in mouse astrocytes by excretory/secretory products of fifth-stage larval Angiostrongylus cantonensis.

BACKGROUND: Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Previous studies have shown that the Sonic hedgehog (Shh) signaling pathway may reduce cell apoptosis by inhibiting oxidative stress in A. cantonensis infection. In this study, we investigated the relationship between cytokine secretion and Shh pathway activation after treatment with excretory/secretory products (ESP) of fifth-stage larval A. cantonensis (L5). RESULTS: The results showed that IL-1ß and IL-6 levels in mouse astrocytes were increased. Moreover, ESP stimulated the protein expression of Shh pathway molecules, including Shh, Ptch, Smo and Gli-1, and induced IL-1ß and IL-6 secretion. The transcription factor nuclear factor-κB (NF-κB) plays an important role in inflammation, and it regulates the expression of proinflammatory genes, including cytokines and chemokines, such as IL-1ß and TNF-α. After ESP treatment, NF-κB induced IL-1ß and IL-6 secretion in astrocytes by activating the Shh signaling pathway. CONCLUSIONS: Overall, the data presented in this study showed that ESP of fifth-stage larval A. cantonensis stimulates astrocyte activation and cytokine generation through NF-κB and the Shh signaling pathway.

Characterization of the N-glycans of female Angiostrongylus cantonensis worms.

Glycoconjugates play a crucial role in the host-parasite relationships of helminthic infections, including angiostrongyliasis. It has previously been shown that the antigenicity of proteins from female Angiostrongylus cantonensis worms may depend on their associated glycan moieties. Here, an N-glycan profile of A. cantonensis is reported. A total soluble extract (TE) was prepared from female A. cantonensis worms and was tested by western blot before and after glycan oxidation or N- and O-glycosidase treatment. The importance of N-glycans for the immunogenicity of A. cantonensis was demonstrated when deglycosylation of the TE with PNGase F completely abrogated IgG recognition. The TE was also fractionated using various lectin columns [Ulex europaeus (UEA), concanavalin A (Con A), Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Lycopersicon esculentum (LEA)], and then each fraction was digested with PNGase F. Released N-glycans were analyzed with matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) and MALDI-TOF/TOF-MS/MS. Complex-type, high mannose, and truncated glycan structures were identified in all five fractions. Sequential MALDI-TOF-TOF analysis of the major MS peaks identified complex-type structures, with a α1-6 fucosylated core and truncated antennas. Glycoproteins in the TE were labeled with BodipyAF558-SE dye for a lectin microarray analysis. Fluorescent images were analyzed with ProScanArray imaging software followed by statistical analysis. A total of 29 lectins showed positive binding to the TE. Of these, Bandeiraea simplicifolia (BS-I), PNA, and Wisteria floribunda (WFA), which recognize galactose (Gal) and N-acetylgalactosamine (GalNAc), exhibited high affinity binding. Taken together, our findings demonstrate that female A. cantonensis worms have characteristic helminth N-glycans.

AcCystatin, an immunoregulatory molecule from Angiostrongylus cantonensis, ameliorates the asthmatic response in an aluminium hydroxide/ovalbumin-induced rat model of asthma.

Epidemiological surveys have demonstrated that helminth infections are negatively related to atopic diseases, including asthma. Defining and characterising specific helminth molecules that have excellent immunomodulatory capacities as potential therapeutics for the treatment or prophylaxis of allergic manifestations are of great interest. AcCystatin, a cystatin protease inhibitor of Angiostrongylus cantonensis, is a homologue of other nematode cystatins with immunoregulatory properties. Here, we aim to determine the effects of AcCystatin on an ovalbumin/aluminium hydroxide (OVA/Al[OH]3)-induced rat model of asthma. Wistar rats were randomly divided into four groups, including a control group, an OVA/Al[OH]3-induced asthma group, a group receiving AcCystatin immunisation prior to OVA/Al[OH]3-induced asthma and a group receiving AcCystatin treatment after OVA/Al[OH]3-induced asthma. The numbers of eosinophils, basophils, neutrophils, lymphocytes and monocytes in the peripheral blood and of eosinophils in the bronchoalveolar lavage fluid (BALF) were counted for each animal. The expression levels of the cytokines interferon-γ, interleukin (IL) 4, IL-5, IL-6, IL-10, IL17A and tumour necrosis factor receptor-α in BALF, of OVA-specific immunoglobulin E in BALF and serum and of the chemokines eotaxin-1, eotaxin-2, eotaxin-3, MCP-1 and MCP-3 in lung tissue were measured. In addition, the degree of peribronchial and perivascular inflammation and the intensity of goblet cell metaplasia were qualitatively evaluated. The sensitised/challenged rats developed an extensive cell inflammatory response of the airways. AcCystatin administration significantly reduced the cellular infiltrate in the perivascular and peribronchial lung tissues and reduced both goblet mucous production and eosinophil infiltration. The rats that were treated with AcCystatin before or after sensitisation with OVA showed significant decreases in eotaxin-1, eotaxin-3 and MCP-1 expression in the lung tissue. The production of IL-4, IL-5, IL-6 and IL-17A and of OVA-specific IgE antibodies was also significantly reduced in AcCystatin-treated rats compared with untreated asthmatic rats. The AcCystatin treatment was associated with a significant increase in IL-10 levels. Our present findings provide the first demonstration that AcCystatin is an effective agent in the prevention and treatment of the airway inflammation associated with asthma.

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis.

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.

Molecular characterization and immunolocalization of a protein disulfide isomerase from Angiostrongylus cantonensis.

Protein disulfide isomerases (PDIs), belonging to the thioredoxin superfamily, are oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds among cysteine residues of proteins. In this study, we report the cloning and characterization of a cDNA encoding a protein disulfide isomerase (AcPDI) from a cDNA library of fourth-stage larvae of Angiostrongylus cantonensis. The deduced amino acid sequence contains two thioredoxin domains and exhibits high identity to the homologues from other species. Quantitative real-time PCR (qRT-PCR) was performed at the third-stage larvae, fourth-stage larvae, and adult stage of A. cantonensis, and the results revealed that the AcPDI mRNA, while expressed at all three stages, is expressed at a significantly higher level in female adult worms. Results of immunohistochemical studies indicated that the AcPDI expression was specifically localized in the tegument and uterus wall of female adult worms. Biochemical analysis showed that recombinant AcPDI was biologically active in vitro and exhibited the typical biochemical functions of PDIs: oxidase/isomerase and reductase activities. Collectively, these results implied that AcPDI may be a female-enriched protein and associated with the reproductive development of A. cantonensis. In addition, considering its biochemical properties, AcPDI may be involved in the formation of the cuticle of A. cantonensis.

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets.

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.