RESUMEN
BACKGROUND: Angiostrongylus cantonensis is a parasite that mainly infects the heart and pulmonary arteries of rats and causes human eosinophilic meningitis or meningoencephalitis in certain geographical areas. Current diagnostic methods include detection of the parasite in cerebrospinal fluid (CSF) and eosinophilic immune examination after lumbar puncture, which may be risky and produce false-positive results. 18F- Fluorodeoxyglucose (FDG), a Positron emission tomography (PET) tracer, has been used to assess different pathological or inflammatory changes in the brains of patients. In this study, we hypothesized that A. cantonensis infection-induced inflammatory and immunomodulatory factors of eosinophils result in localized pathological changes in the brains of non-permissive hosts, which could be analyzed using in vivo 18F-FDG PET imaging. METHODOLOGY/FINDINGS: Non-permissive host ICR mice and permissive host SD rats were infected with A. cantonensis, and the effects of the resulting inflammation on 18F-FDG uptake were characterized using PET imaging. We also quantitatively measured the distributed uptake values of different brain regions to build an evaluated imaging model of localized neuropathological damage caused by eosinophilic inflammation. Our results showed that the uptake of 18F-FDG increased in the cerebellum, brainstem, and limbic system of mice at three weeks post-infection, whereas the uptake in the rat brain was not significant. Immunohistochemical staining and western blotting revealed that Iba-1, a microglia-specific marker, significantly increased in the hippocampus and its surrounding area in mice after three weeks of infection, and then became pronounced after four weeks of infection; while YM-1, an eosinophilic chemotactic factor, in the hippocampus and midbrain, increased significantly from two weeks post-infection, sharply escalated after three weeks of infection, and peaked after four weeks of infection. Cytometric bead array (CBA) analysis revealed that the expression of TNF in the serum of mice increased concomitantly with the prolongation of infection duration. Furthermore, IFN-γ and IL-4 in rat serum were significantly higher than in mouse serum at two weeks post-infection, indicating significantly different immune responses in the brains of rats and mice. We suggest that 18F-FDG uptake in the host brain may be attributed to the accumulation of large numbers of immune cells, especially the metabolic burst of activated eosinophils, which are attracted to and induced by activated microglia in the brain. CONCLUSIONS: An in vivo 18F-FDG/PET imaging model can be used to evaluate live neuroinflammatory pathological changes in the brains of A. cantonensis-infected mice and rats.
Asunto(s)
Angiostrongylus cantonensis , Encéfalo , Fluorodesoxiglucosa F18 , Tomografía de Emisión de Positrones , Ratas Sprague-Dawley , Infecciones por Strongylida , Animales , Angiostrongylus cantonensis/inmunología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/diagnóstico por imagen , Infecciones por Strongylida/patología , Encéfalo/parasitología , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Encéfalo/inmunología , Ratones , Ratas , Eosinófilos/inmunología , Inflamación/inmunología , Masculino , Modelos Animales de Enfermedad , Lectinas/metabolismo , Femenino , beta-N-AcetilhexosaminidasasRESUMEN
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.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Meningoencefalitis/diagnóstico , Meningoencefalitis/parasitología , Infecciones por Strongylida/diagnóstico , Secuencia de Aminoácidos , Angiostrongylus cantonensis/química , Animales , Antígenos Helmínticos/sangre , Antígenos Helmínticos/química , Antígenos Helmínticos/aislamiento & purificación , Western Blotting , Secuencia Conservada , Reacciones Cruzadas , Electroforesis , Electroforesis en Gel Bidimensional , Proteínas del Helminto/química , Proteínas del Helminto/aislamiento & purificación , Humanos , Inmunoensayo , Pruebas Inmunológicas , Espectrometría de Masas , Meningoencefalitis/inmunología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitologíaRESUMEN
Angiostrongyliasis is induced by the nematode Angiostrongylus cantonensis and leads to eosinophilic meningitis and meningoencephalitis in humans. Excretory-secretory products (ESPs) are important investigation targets for studying the relationship between hosts and nematodes. These products assist worms in penetrating the blood-brain barrier and avoiding the host immune response. Autophagy is a catabolic process that is responsible for digesting cytoplasmic organelles, proteins, and lipids and removing them through lysosomes. This process is essential to cell survival and homeostasis during nutritional deficiency, cell injury and stress. In this study, we investigated autophagy induction upon treatment with the ESPs of the fifth-stage larvae (L5) of A. cantonensis and observed the relationship between autophagy and the Shh pathway. First, the results showed that A. cantonensis infection induced blood-brain barrier dysfunction and pathological changes in the brain. Moreover, A. cantonensis L5 ESPs stimulated autophagosome formation and the expression of autophagy molecules, such as LC3B, Beclin, and p62. The data showed that upon ESPs treatment, rapamycin elevated cell viability through the activation of the autophagy mechanism in astrocytes. Finally, we found that ESPs induced the activation of the Sonic hedgehog (Shh) signaling pathway and that the expression of autophagy molecules was increased through the Shh signaling pathway. Collectively, these results suggest that A. cantonensis L5 ESPs stimulate autophagy through the Shh signaling pathway and that autophagy has a protective effect in astrocytes.
Asunto(s)
Angiostrongylus cantonensis/metabolismo , Astrocitos/parasitología , Autofagia , Encéfalo/patología , Proteínas Hedgehog/metabolismo , Transducción de Señal , Angiostrongylus cantonensis/inmunología , Animales , Astrocitos/citología , Barrera Hematoencefálica/fisiopatología , Encéfalo/parasitología , Interacciones Huésped-Parásitos , Larva/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , CaracolesRESUMEN
Cerebral angiostrongyliasis is a central nervous system disease caused by Angiostrongylus cantonensis and can produce eosinophilic meningitis or meningoencephalitis in humans. Sero-immunological techniques, such as the enzyme-linked immunosorbent assay (ELISA) and immunoblotting, are most commonly used for the diagnosis of human angiostrongyliasis. However, diagnosis in remote areas remains problematic because sophisticated equipment and specialized skills are required. To overcome, we have developed the immunochromatographic test (ICT) kit for rapid serological diagnosis of angiostrongyliasis through the detection of anti-A. cantonensis-specific antibodies in human serum. A recombinant A. cantonensis galectin-2 (rAcGal2) from young adult female worms was used as an antigen for the ICT kit development. Diagnostic values were evaluated and compared using the ELISA. The sensitivity, specificity and positive and negative predictive values of the ICT kit were 87.0, 96.5, 94.6 and 91.4%, respectively, and those of the ELISA were 91.0, 97.2, 95.8 and 94.0%, respectively. The concordance of the ICT kit was 93.9%. We, thus, determined that the ICT kit is sensitive and specific and provides reliable diagnostic results. It is rapid and simple to perform and can be utilized for both point-of-care diagnosis in the bedside laboratory and epidemiological surveys in endemic regions where access to diagnostic equipment is limited.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Anticuerpos Antihelmínticos/sangre , Inmunoensayo/métodos , Infecciones por Strongylida/diagnóstico , Animales , Antígenos Helmínticos/inmunología , Femenino , Proteínas del Helminto/inmunología , Humanos , Valor Predictivo de las Pruebas , Juego de Reactivos para Diagnóstico/normas , Sensibilidad y Especificidad , Infecciones por Strongylida/parasitologíaRESUMEN
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.
Asunto(s)
Angiostrongylus cantonensis/química , Angiostrongylus cantonensis/inmunología , Quimiotaxis/inmunología , Eosinófilos/inmunología , Galectinas/líquido cefalorraquídeo , Animales , Western Blotting , Encéfalo/inmunología , Eosinófilos/fisiología , Femenino , Galectinas/inmunología , Estadios del Ciclo de Vida , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB CRESUMEN
Angiostrongyliasis is a foodborne disease caused by a zoonotic nematode, Angiostrongylus cantonensis, which produces eosinophilic meningitis or meningoencephalitis (EOM) in humans. Definitive diagnosis is rarely possible because worms are almost never recovered from patients. Human disease can be diagnosed by clinical symptoms and serological tests. Presently, diagnosis is performed by serological detection of antibodies against specific somatic antigens (molecular mass 29-31 kDa) extracted from female worms. The life cycle of A. cantonensis must be maintained in the laboratory to provide a source of this diagnostic antigen. Here, we cloned and expressed recombinant A. cantonensis galectin-2 (rAcGal2) corresponding to a 31-kDa antigenic peptide. Recombinant protein was purified and used in immunoblot tests, which showed reactions with human serum panels consisting of six confirmed angiostrongyliasis and 24 clinically diagnosed cases of EOM-associated with angiostrongyliasis, 160 samples from patients with other parasitic infections, and 30 samples from normal healthy subjects. Accuracy, sensitivity, specificity, and positive and negative predictive values were 95.0%, 93.3%, 95.3%, 75.7%, and 98.9%, respectively. The test was nonreactive with sera of human gnathostomiasis and cysticercosis, two diseases that could present similar neurological symptoms. Recombinant AcGal2 has potential as a diagnostic antigen and could replace native parasite antigens in further development of an angiostrongyliasis serodiagnostic test kit.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Anticuerpos Antihelmínticos/sangre , Galectina 2/inmunología , Proteínas Recombinantes/inmunología , Infecciones por Strongylida/diagnóstico , Animales , Antígenos Helmínticos/inmunología , Humanos , Immunoblotting , Pruebas SerológicasRESUMEN
BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) is a foodborne parasite that can invade the central nervous system (CNS), resulting in eosinophilic meningitis (EM). However, the mechanism by which A. cantonensis causes eosinophilic infiltration into CNS is not well understood. METHODS: In this study eosinophilic infiltration into the CNS caused by A. cantonensis was assessed based on eosinophil counts and evaluation of interleukin (IL)-5 and -13 levels by real-time PCR in brain of Balb/c mice. The expression and activation of IL-17A, IL17 receptor (IL-17R A), and IL-17RC and the related signaling molecules nuclear factor (NF)-κB1, NF-κB2, NF-κB activator (Act)1, tumor necrosis factor receptor-associated factor (Traf)5, and Traf6 during A. cantonensis infection in brain tissue of Balb/c mice were examined by real-time, western blotting and immunofluroence. A. cantonensis-infected Balb/c mice were treated with IL-17A neutralizing antibody to evaluate the role of IL17A in eosinophil accumulation in the CNS. RESULTS: Our results showed A. cantonensis infection caused eosinophil accumulation and alterations in IL-5 and -13 levels. The expression of IL-17A and -17RA, Act1, and Traf6 but not of IL-17RC and Traf5 was upregulated during infection; this was accompanied by NF-κB1 and -κB2 activation. Importantly, application of IL-17A neutralizing antibody attenuated eosinophil accumulation in CNS and reversed the changes in IL-5 and -13 expression caused by A. cantonensis infection. Additionally, IL-17RA and Traf6 levels decreased, which was accompanied by NF-κB inactivation. CONCLUSION: IL-17A plays an important role in EM caused by A. cantonensis, possibly through activation of NF-κB via the IL-17RA/Traf6 signaling pathway. These findings highlight the potential for using IL-17A neutralizing antibody as a therapeutic strategy for the treatment of EM.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Anticuerpos Neutralizantes/inmunología , Eosinófilos/inmunología , Interleucina-17/inmunología , Meningitis/inmunología , FN-kappa B/inmunología , Receptores de Interleucina-17/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Animales , Encéfalo/inmunología , Encéfalo/parasitología , Citocinas/inmunología , Eosinófilos/parasitología , Meningitis/parasitología , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/inmunología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Activación Transcripcional/inmunologíaRESUMEN
BACKGROUND: To determine whether intestinal Angiostrongylus cantonensis antigens can induce protective immunity in rats, gut antigens prepared from female adults (FAGP) and somatic antigens prepared from both male (MA) and female (FA) adult worms were used to immunize rats. METHODS: Rats were immunized twice with MA, FA, or FAGP antigens and then challenged with 50 third-stage A. cantonensis larvae, and different readouts were used to monitor protective immunity. Additionally, protein profiles of MA, FA, and FAGP extracts were analyzed and characterized by immunodetection methods. RESULTS: A 15% reduction in fifth-stage larvae from brains and a 14% reduction in adult worms from pulmonary arteries were observed in rats immunized with FAGP compared to controls. However, there was a >50% reduction in rats immunized with MA or FA. The lengths of larvae and adults recovered from FAGP-immunized rats were shorter than those recovered from other groups. The number of first-stage larvae recovered from fecal material in FAGP-immunized rats was significantly reduced. Additionally, FAGP induced the highest splenocyte proliferation. Serum IgG titers were not directly correlated with protective immunity. An 84 kDa gut membrane protein was strongly recognized by anti-FAGP antibodies. CONCLUSION: We demonstrated that immune responses induced by FAGP reduced the growth, development, and reproduction of A. cantonensis in subsequent infections. While the possibility of using FAGP combining with MA or FA antigens as a multi-function vaccine in immune protection against A. cantonensis needs to be further elucidated, we hope that it provides a novel strategy for this parasite vaccine development.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Intestinos , Proteínas/inmunología , Animales , Anticuerpos Antihelmínticos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Femenino , Inmunización , Inmunoglobulina G/sangre , Masculino , Proteínas de la Membrana/inmunología , Ratas , Ratas Sprague-Dawley , Vacunación , Vacunas AnticonceptivasRESUMEN
OBJECTIVES: The serological diagnosis of human infection with Angiostrongylus cantonensis remains problematic because there are no commercially available validated tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or immunoblotting. Since laboratory facilities are not always available in endemic areas, we developed and assessed a rapid lateral flow immunochromatographic assay (AcQuickDx Test) to detect anti-A. cantonensis antibodies in human serum. METHODS: The test device was assembled with purified 31-kDa glycoprotein as diagnostic antigen and with gold-labelled anti-human immunoglublin-G as the detector reagent. A total of 97 serum samples were tested - 19 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 43 samples from patients with other parasitic diseases, i.e. gnathostomiasis (n=13), toxocariasis (n=2), trichinellosis (n=2), hookworm infection (n=4), filariasis (n=5), cysticercosis (n=9), paragonimiasis (n=2), opisthorchiasis (n=3), and malaria (n=3); and 35 samples from normal healthy subjects. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of AcQuickDx Test to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were 100%, 98.72%, 95% and 100%, respectively. Positive AcQuickDx was observed in 1 of 4 cases with hookworm infections. No positive AcQuickDx was observed in cases with other parasitic diseases, and the individual healthy subjects. CONCLUSIONS: AcQuickDx Test is rapid, highly sensitive and specific, and easy to perform without additional equipment or ancillary supplies. It yields results that are interpreted visually, and possesses a long shelf-life at room temperature. Thus, it can be applied as an additional test for clinical diagnostic support of angiostrongyliasis either in conventional laboratories or for remote areas where laboratory infrastructure is not available.
Asunto(s)
Cromatografía de Afinidad/métodos , Infecciones por Strongylida/diagnóstico , Angiostrongylus cantonensis/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Humanos , Pruebas SerológicasRESUMEN
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.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Galectina 1/inmunología , Proteínas del Helminto/inmunología , Infecciones por Strongylida/inmunología , Angiostrongylus cantonensis/química , Angiostrongylus cantonensis/patogenicidad , Animales , Encéfalo/parasitología , Encéfalo/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Galectina 1/metabolismo , Proteínas del Helminto/metabolismo , Humanos , Inmunidad Celular , Inmunidad Humoral , Ratones , Ratones Endogámicos C57BL , Organismos Libres de Patógenos Específicos , Bazo/parasitología , Bazo/patología , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/patología , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: The parasitic nematode Angiostrongylus cantonensis is the primary pathogen causing eosinophilic meningitis and meningoencephalitis in nonpermissive hosts. The larval parasites are eliminated by the host's immune responses in the central nervous system (CNS) through infiltration of eosinophils and lymphocytes. This study aimed to determine primary alterations of microRNA (miRNA) during A. cantonensis infection in mice. METHODS: miRNA array was used to analyze the expression of miRNA in uninfected and A. cantonensis-infected mouse brains at 21 days postinfection (dpi). Target genes were predicted by miRDB software, and protein-protein interaction network was analyzed using STRING v9.1. Expression levels of selected miRNAs and cytokine production were verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Twenty-five mature miRNAs showed differential expression in infected mouse brains, of which 24 were upregulated and one was downregulated compared to the uninfected control. These 25 miRNAs were divided into five clusters, and the first upregulated cluster was selected for further bioinformatics analysis. Target gene prediction and gene ontology (GO) enrichment analysis revealed that the miRNAs were mainly related to the immune response. Furthermore, six target genes of mmu-miR-146a-5p were predicted to interact with tumor necrosis factor alpha (TNF-α). The in vitro study suggested that transfected mmu-miR-146a-5p inhibitor upregulated TNF-α and its target gene Traf6 in microglia following stimulation with A. cantonensis larval antigen. CONCLUSION: This study suggested a critical role of miRNAs in the host defense during A. cantonensis infection, providing new insights into the molecular mechanisms underlying the interaction between mmu-miR-146a-5p and TNF-α in angiostrongyliasis in nonpermissive hosts.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Angiostrongylus cantonensis/patogenicidad , Encéfalo/metabolismo , Encéfalo/parasitología , MicroARNs/biosíntesis , Inmunidad Adaptativa , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/parasitología , Análisis por Conglomerados , Biología Computacional , Citocinas/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Interacciones Huésped-Parásitos/inmunología , Larva/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Microglía , Dominios y Motivos de Interacción de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of eosinophilic meningoencephalitis worldwide. This parasite is endemic to Southeast Asia and the Pacific Islands, and its global distribution is increasing. We report A. cantonensis meningoencephalitis in a 12-month-old boy in Tennessee, USA, who had not traveled outside of southwestern Tennessee or northwestern Mississippi.
Asunto(s)
Angiostrongylus cantonensis/aislamiento & purificación , Eosinofilia/diagnóstico , Meningoencefalitis/diagnóstico , Infecciones por Strongylida/diagnóstico , Corticoesteroides/uso terapéutico , Albendazol/uso terapéutico , Angiostrongylus cantonensis/efectos de los fármacos , Angiostrongylus cantonensis/inmunología , Angiostrongylus cantonensis/patogenicidad , Animales , Anticuerpos Antihelmínticos/sangre , Eosinofilia/tratamiento farmacológico , Eosinofilia/parasitología , Humanos , Lactante , Masculino , Meningoencefalitis/tratamiento farmacológico , Meningoencefalitis/parasitología , Ratas , Infecciones por Strongylida/tratamiento farmacológico , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/transmisiónRESUMEN
The nematode Angiostrongylus cantonensis (A.C.) is a neurotropic pathogen; stage-III larva invade the human (non-permissive host) central nervous system (CNS) to cause eosinophilic meningitis or meningoencephalitis accompanied by immunosuppression. In an A.C.-infectedmouse (another non-permissive host) model, CNS damage-associated T cell immune deficiency and severe inflammation were proposed to result from activation of the hypothalamic-pituitary-adrenal (HPA) axis. However, glucocorticoids are anti-inflammatory agents. Additionally, while defects in thymic stromal/epithelial cells (TECs) are the major reason for thymic atrophy, TECs do not express the glucocorticoid receptor. Therefore, activation of the HPA axis cannot fully explain the thymic atrophy and inflammation. Using an A.C.-infected mouse model, we found that A.C.-infected mice developed severe thymic atrophy with dramatic impairments in thymocytes and TECs, particularly cortical TECs, which harbor CD4+CD8+ double-positive thymocytes. The impairments resulted from soluble antigens (sAgs) from A.C. in the thymuses of infected mice, as intrathymic injection of these sAgs into live mice and the addition of these sAgs to thymic cell culture resulted in thymic atrophy and cellular apoptosis, respectively. Therefore, in addition to an indirect effect on thymocytes through the HPA axis, our study reveals a novel mechanism by which A.C. infection in non-permissive hosts directly induces defects in both thymocytes and TECs via soluble antigens.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Infecciones por Strongylida/inmunología , Timo/inmunología , Timo/patología , Animales , Apoptosis/inmunología , Atrofia , Modelos Animales de Enfermedad , Masculino , Ratones , Células del Estroma/metabolismo , Infecciones por Strongylida/parasitología , Timocitos/inmunología , Timocitos/metabolismoRESUMEN
Angiostrongylus cantonensis, the rat lungworm, is an important aetiologic agent of eosinophilic meningitis and meningoencephalitis in humans. Co-culturing astrocytes with soluble antigens of A. cantonensis activated the Sonic hedgehog (Shh) signalling pathway and inhibited the apoptosis of astrocytes via the activation of Bcl-2. This study was conducted to determine the roles of the Shh signalling pathway, apoptosis, and oxidative stress in astrocytes after treatment with excretory-secretory products (ESP) from A. cantonensis fifth-stage larvae. Although astrocyte viability was significantly decreased after ESP treatment, the expression of Shh signalling pathway related proteins (Shh, Ptch-1 and Gli-1) was significantly increased. However, apoptosis in astrocytes was significantly decreased after activation of the Shh signalling pathway. Moreover, superoxide and hydrogen superoxide levels in astrocytes were significantly reduced after the activation of Shh pathway signalling due to increasing levels of the antioxidants catalase and superoxide dismutase. These findings indicate that the anti-apoptotic effects of the Shh signalling pathway in the astrocytes of mice infected with A. cantonensis are due to reduced levels of oxidative stress caused by the activation of antioxidants.
Asunto(s)
Angiostrongylus cantonensis/metabolismo , Antígenos Helmínticos/metabolismo , Apoptosis , Astrocitos/metabolismo , Proteínas Hedgehog/metabolismo , Estrés Oxidativo , Transducción de Señal , Angiostrongylus cantonensis/inmunología , Animales , Antígenos Helmínticos/inmunología , Técnicas de Cocultivo , Interacciones Huésped-Parásitos , Larva , Ratones , Ratas , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
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.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Meningoencefalitis/diagnóstico , Meningoencefalitis/parasitología , Infecciones por Strongylida/diagnóstico , Angiostrongylus cantonensis/metabolismo , Animales , Reacciones Cruzadas , Humanos , Meningoencefalitis/sangre , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitologíaRESUMEN
The spleen is one of the most important peripheral immune organs, which is frequently affected in infectious diseases. Infectious diseases can induce splenic alterations including splenic atrophy and functional alteration, while splenic atrophy may in turn interferes with recovery of infectious diseases. Angiostrongyliasis is an infectious disease by Angiostrongylus cantonensis (A. cantonensis), which invade non-permissive hosts, such as humans and mice, to cause severe damage to the central nervous system (CNS) and acute inflammatory response. A. cantonensis infection-induced CNS injury has been confirmed to be due to profound immunopathology derived from peripheral immune components. However, the mechanism of immunopathology remains largely unknown. Here, we found that A. cantonensis invaded non-permissive hosts such as mice in the brain, but not in the other peripheral organs. However, this infection induced severe spleen atrophy. We further recognized that this atrophy is associated with a decrease of total splenocyte number and disruption of splenic structure due to reduced proliferation and increased apoptotosis. These also resulted in deterioration of T cell profile in the periphery with a low CD4/CD8 ratio and B/T cell ratio, and increased ratio of CD4+CD25+Foxp3+ Treg, CD8+CD28- T, and CD38+T lymphocyte of spleen. Albendazole treatment can alleviate spleen atrophy and set T cell immune reconstitution in some extend. Our data showed that A. cantonensis infection can cause splenic atrophy. These results are suggested to put more emphasis to improve the function of immune system. Meanwhile, infection and treatment model will be useful to evaluate new therapeutic approaches which can prevent or reverse immunosuppression and infectious complications.
Asunto(s)
Angiostrongylus cantonensis/fisiología , Bazo/inmunología , Infecciones por Strongylida/inmunología , Angiostrongylus cantonensis/inmunología , Animales , Biomphalaria/parasitología , Encéfalo/inmunología , Encéfalo/parasitología , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Bazo/parasitología , Bazo/patología , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/patología , Linfocitos T Reguladores/inmunologíaRESUMEN
Angistrongylus cantonensis is a zoonotic nematode parasite and causative agent of human angiostrongyliasis, which clinically presents as eosinophilic meningitis or meningoencephalitis. Diagnosis of the disease is problematic since parasitologic findings are infrequent, and infection determinations must be based on the clinical symptoms and serological tests with limited specificities and sensitivities. The aim of the present study was to identify and generate a novel recombinant protein from A. cantonensis and evaluate its efficacy in the diagnosis of human angiostrongyliasis when incorporated into a Western blot serodiagnostic system. A cDNA protein expression library from adult A. cantonensis was constructed, followed by immunoscreening with serum from confirmed infected patients to identify and isolate immunoreactive clones. One clone, designated fAC40, possessed a partial sequence encoding a LisH protein domain with a predicted molecular weight of 16 kDa and containing four predicted antigenic peptides. By incorporating recombinant fAC40 in Western immunoblot tests using a serum panel consisting of confirmed and clinically diagnosed cases of human angiostrongyliasis and other helminthic infections, fAC40 exhibited a sensitivity and specificity of 91.8 and 100 %, respectively, and a positive and negative predictive value of 100 and 97.19 %, respectively, in the diagnosis of angiostrongyliasis. Importantly, it was not reactive with antibodies from serum of patients infected with Gnathostoma spinigerum and Cysticercus cellulosae, infections that clinically present neurological symptoms similar to angiostrongyliasis. These data demonstrate that the 16-kDa recombinant protein from A. cantonensis possesses high potential as a candidate antigen for a more sensitive and specific serodiagnosis of human angiostrongyliasis.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Meningoencefalitis/diagnóstico , Infecciones por Strongylida/diagnóstico , Adulto , Secuencia de Aminoácidos , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/aislamiento & purificación , Animales , Antígenos Helmínticos/genética , Secuencia de Bases , Western Blotting , Cisticercosis/diagnóstico , Cisticercosis/parasitología , Cysticercus/inmunología , Cysticercus/aislamiento & purificación , Femenino , Gnathostoma/inmunología , Gnathostoma/aislamiento & purificación , Gnathostomiasis/diagnóstico , Gnathostomiasis/parasitología , Proteínas del Helminto/genética , Humanos , Immunoblotting , Meningoencefalitis/parasitología , Proteínas Recombinantes , Sensibilidad y Especificidad , Infecciones por Strongylida/parasitologíaRESUMEN
Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.
Asunto(s)
Angiostrongylus cantonensis/aislamiento & purificación , Antígenos Helmínticos/análisis , Cromatografía de Afinidad/métodos , Pruebas Diagnósticas de Rutina/métodos , Infecciones por Strongylida/diagnóstico , Adulto , Angiostrongylus cantonensis/inmunología , Animales , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.
Asunto(s)
Angiostrongylus/patogenicidad , Modelos Animales de Enfermedad , Eosinofilia/parasitología , Meningitis/parasitología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Angiostrongylus/inmunología , Angiostrongylus cantonensis/inmunología , Angiostrongylus cantonensis/patogenicidad , Animales , Antígenos Helmínticos/sangre , Antígenos Helmínticos/inmunología , Encéfalo/parasitología , Encéfalo/patología , Citocinas/biosíntesis , Citocinas/inmunología , Eosinofilia/inmunología , Femenino , Cobayas , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Meningitis/inmunología , Ratones , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
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.