Exacerbation of allergic asthma by somatic antigen of Echinococcus granulosus in allergic airway inflammation in BALB/c mice.

BACKGROUND: There is ample evidence demonstrating a reverse relationship between helminth infection and immune-mediated diseases. Accordingly, several studies have shown that Echinococcus granulosus infection and hydatid cyst compounds are able to suppress immune responses in allergic airway inflammation. Previous studies have documented the ability of hydatid cysts to suppress aberrant Th2 immune response in a mouse model of allergic asthma. However, there is a paucity of research on the effects of protoscoleces on allergic asthma. Thus, this study was designed to evaluate the effects of somatic antigens of protoscoleces in a murine model of allergic airway inflammation. METHODS: Ovalbumin (OVA)/aluminum hydroxide (alum) was injected intraperitoneally to sensitize BALB/c mice over a period of 0 to 7 days, followed by challenge with 1% OVA. The treatment group received somatic antigens of protoscoleces emulsified with PBS on these days in each sensitization before being challenged with 1% OVA on days 14, 15, and 16. The effects of somatic antigens of protoscoleces on allergic airway inflammation were evaluated by examining histopathological changes, the recruitment of inflammatory cells in the bronchoalveolar lavage, cytokine production in the homogenized lung tissue (IL-4, IL-5, IL-10, IL-17, and IFN-γ), and total antioxidant capacity in serum. RESULTS: Overall, administration of somatic antigens of protoscoleces exacerbated allergic airway inflammation via increased Th2 cytokine levels in the lung homogenate, recruitment of eosinophils into bronchoalveolar lavage fluid, and pathological changes. In addition, total antioxidant capacity and IFN-γ levels declined following the administration of somatic antigens. CONCLUSIONS: The results revealed that the co-administration of somatic products of protoscoleces with OVA/alum contributed to the exacerbation of allergic airway inflammation in BALB/c mice. Currently, the main cause of allergic-type inflammation exacerbation is unknown, and further research is needed to understand the mechanism of these interactions.

Fasciola hepatica coinfection modifies the morphological and immunological features of Echinococcus granulosus cysts in cattle.

Polyparasitism occurs when animals harbour multiple parasites concomitantly. It is a common occurrence but is generally understudied in wild and domestic animals. Fasciola hepatica and Echinococcus granulosus, which are helminths of ungulates, frequently coinfect cattle. The effects of this particular type of polyparasitism are not well documented. The metacestode of Echinococcus granulosus is surrounded by the adventitial layer, which constitutes the host immune response to the parasite. This layer in cattle is produced by a granulomatous reaction and is involved in echinococcal cyst (EC) fertility. Due to the systemic immune-modulating abilities of Fasciola hepatica, coinfection possibly generates a favourable environment for EC growth. A total of 203 Echinococcus granulosus sensu stricto cysts were found in 82 cattle, of which 42 ECs were found in 31 animals coinfected with Fasciola hepatica. The overall infection intensity was 3 cysts per animal. Coinfection with Fasciola hepatica decreased the mean infection intensity to 1.4 cysts per animal. Regarding EC size, coinfection resulted in smaller ECs (15.91 vs 22.09 mm), especially for infertile lung cysts. The adventitial layer of ECs in coinfected animals lacked lymphoid follicles and palisading macrophages, which are generally hallmarks of the granulomatous immune response. The ECs in coinfected animals had organized laminated layers, whereas those in animals without coinfection did not. Although coinfection was not statistically associated with EC fertility, we did not find fertile cysts in the livers of coinfected animals. We concluded that coinfection with Fasciola hepatica and Echinococcus granulosus has a detrimental effect on ECs, particularly infertile cysts.

[Pulmonary granuloma in an immunodepressed patient]. / Un granulome pulmonaire chez une patiente immunodéprimée.

INTRODUCTION: Pulmonary alveolar echinococcosis is a rare but potentially severe condition. CASE REPORT: We report the case of a 50-year-old woman suffering from pulmonary alveolar echinococcosis who had had a renal transplant for polycystic liver and kidney disease. A lung opacity was identified radiologically in May 2013. Both broncho-alveolar lavage and bronchial biopsy were uninformative. In January 2014, a follow up CT-scan showed the opacity to be enlarging. A surgical biopsy revealed a giant cell epithelioid granuloma with caseous necrosis suggesting a diagnosis of pulmonary tuberculosis. Antituberculous treatment was started but cultures remained negative. A histological revue was therefore requested in March 2014. This suggested bronchocentric granulmatosis, possibly associated with echinococcosis. This hypothesis was finally confirmed serologically. Treatment for alveolar echinococcosis was begun in June 2014 after consultation with the national reference centre for parasitology. CONCLUSION: Outside endemic areas and in the absence of hepatic involvement pulmonary alveolar echinococcosis can be difficult to diagnose. This case report focuses on the diagnostic criteria and treatment.

Serological and molecular characteristics of the first Korean case of Echinococcus multilocularis.

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient's serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.

Comparative analysis of the diagnostic performance of crude sheep hydatid cyst fluid, purified antigen B and its subunit (12 Kda), assessed by ELISA, in the diagnosis of human cystic echinococcosis.

The diagnosis of patients with cystic echinococcosis (CE) by means of serology has a limited support in clinical practice due to cross-reactivity with other helminthes leading to overestimation of the parasite's true prevalence. A wealth of reports on the diagnostic performance of antigen B (AgB) has been produced. This study was designed to comparatively assess the diagnostic efficacy of crude sheep hydatid cyst fluid (HCF), AgB and its subunit (12 KDa) to detect IgG or IgG4 antibodies in CE patients' sera using enzyme-linked immunosorbent assay (ELISA).The best diagnostic performance was obtained with anti-HCF IgG ELISA which gave 92.4% sensitivity and 92.6% specificity. Despite the low sensitivity of anti AgB IgG ELISA (84%), it gave the best specificity (94.4%) with less cross-reaction with sera of subjects infected with other parasites. In conclusion, it is recommended to use anti-HCF IgG ELISA for initial screening in large seroprevalence studies. Further analysis of positive serum samples with anti AgB IgG ELISA would allow the confirmation of true positives. Specific IgG4 ELISA may represent a complementary assay, useful as secondary confirmatory tests for patients with suspected CE and negative for total IgG ELISA.

Immunoglobulin G subclass responses to recombinant Em18 in the follow-up of patients with alveolar echinococcosis in different clinical stages.

In this study, we compared the sequential responses of immunoglobulin G (IgG) subclasses to the diagnostic antigen Em18 in sera from patients with alveolar echinococcosis. A total of 225 sera from 36 patients at different clinical stages according to the WHO-PNM staging system were tested. The antibody responses were measured for cohorts with resected and unresected parasitic lesions by enzyme-linked immunosorbent assays (ELISA). Total IgG and, to a lesser extent, IgG4 antibody levels against Em18 correlated with all PNM stages before treatment, whereas levels of IgG2 were low and IgG3 was undetectable. Antibody kinetics, however, depended on the treatment rather than on the PNM stage. For some patients, after curative surgery, IgG1 antibodies dropped below the cutoff earlier than other antibodies, followed by total IgG and IgG4 within 18 months. For some patients with recurrences after surgery, IgG1 and IgG4 reappeared, whereas patients with unresectable lesions but stable disease showed steady declines in the levels of all antibodies, and IgG1 became undetectable in some patients. Additional testing of IgE responses to Em18 showed constantly low levels at all stages and in all cohorts.

Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis.

Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestode stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-à-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development.

Involvement of IL-10 and IL-4 in evasion strategies of Echinococcus granulosus to host immune response.

Human echinococcosis is one of the world's major zoonotic infections. In this study, our aim is to clarify one of the strategies used by the parasite for evasion and prolonged infestation in the host. We wished to provide further immunological evidence for the involvement of IL-10/IL-4 in these mechanisms. In this regard, we investigated the effects of IL-4 and IL-10 on protoscoleces (PSC: the larval form of the parasite), co-cultured with patient PBMC. Furthermore, we used IL-4 and IL-10 antibodies to confirm this effect. Our results showed that IL-4 and IL-10 reduced PSC killing. This reduction correlates with an decrease in NO production by PBMC. In conclusion, the results reported here suggest that IL-4/IL-10 impairs the Th1 protective response and allows the parasite to survive in hydatid patients.

Immune response of mice with alveolar echinococcosis to therapy with transfer factor, alone and in combination with albendazole.

The effect of dialysable leucocyte extract (transfer factor TF) on immune response of mice infected with Echinococcus multilocularis and treated with albendazole (ABZ) was observed. TF administration increased the parasite-suppressed proliferative response of T and B lymphocytes of infected mice from weeks 8 to 12 or 14 post infection (p.i.), respectively, with the most stimulative effect after TF+ABZ therapy. The CD4 T cell presence in the spleen of infected mice with TF or TF+ABZ therapy was increased from weeks 6 to 12 or 14 p.i., respectively. The production of IFN-gamma (Th1 cytokine) after TF or TF+ABZ therapy was significantly higher from weeks 6 to 12 p.i., and during this time, the significantly inhibited IL-5 synthesis (Th2 cytokine) was detected, particularly after TF+ABZ therapy. The superoxide anion (O2-) production in peritoneal macrophages of infected mice treated with TF or TF+ABZ was stimulated from weeks 8 to 18 p.i. The immunomodulative effect of TF reduced the growth of larval cysts till week 14 p.i. with a comparable intensity to the anthelmintic drug ABZ. Combined therapy TF+ABZ resulted in the greatest parasite restriction and reduced the cyst development till the end of the experiment.

Serological confirmatory testing of alveolar and cystic echinococcosis in clinical practice: results of a comparative study with commercialized and in-house assays.

Sera of 50 patients with either cystic (CE) or alveolar echinococcosis (AE) in different clinical stages were examined for the presence of anti-Echinococcus-antibodies. Antibody-screening was performed with ELISA, IHA and IFAT, and confirmatory testing was done by the commercialized E. multilocularis-specific Em2plus-ELISA versus an in-house E. multilocularis-specific Em10-ELISA. Sera with discrepant confirmatory results were subjected to a commercial Echinococcus IgG Western blot (WB). In sera from patients with CE, the Em2plus-ELISA showed cross-reactions in 23.5%, whereas the Em10-ELISA did not exhibit any cross-reactivity. Cross-reactivity paralleled active infection with high antibody titers in the screening assays. In sera from patients with AE, confirmation by both ELISAs was achieved in 57.6%, mostly in patients with an advanced stage of the disease and high antibody titers in the screening assays. False-negative reactions of both ELISAs occurred in 30.3%, mostly in patients who had low antibody levels in the screening tests. The Em2plus-ELISA exhibited fewer false-negative reactions than the Em10-ELISA. The WB confirmed the positive results of either assay and was the assay with the highest reliability at different stages of CE and AE, followed by the Em2plus-ELISA for AE. High antibody titers in the screening assays will favour the detection of species-specific antibodies in either form.

Evaluation of a commercial Echinococcus Western Blot assay for serological follow-up of patients with alveolar echinococcosis.

A total of 20 patients with alveolar echinococcosis in different clinical stages according to the WHO-PNM staging system (P, parasitic mass in the liver; N, involvement of neighboring organs; M, metastasis) were followed up serologically with the commercial Echinococcus Western Blot IgG assay and a crude antigen extract enzyme-linked immunosorbent assay (ELISA). The cohort included patients after curative resection and patients who had unresectable lesions with stable disease or progressive infection. There were visible correlations of the crude antigen ELISA index and the presence and intensity of diagnostic bands in the Western blot. In most patients after curative resection, bands at 7, 16, and 18 kDa markedly decreased or vanished after 1 to 4 years. In a patient with a nonviable lesion (it died out), bands at 16 and 18 kDa vanished after 4 years. Among individuals with unresectable lesions but stable disease under antiparasitic chemotherapy, a decrease of all diagnostic bands was visible after 2 to 3 years in half of the patients, whereas the other half had unchanged blot results after 4 to 6 years. Patients with progressive disease showed increasing intensities of bands at 16, 18, and 7 kDa. The change of banding patterns was not influenced by the PNM stage in patients after curative surgery or with unresectable lesions. Our data indicate a correlation of the 7-, 16-, and 18-kDa-Western blot bands with disease activity independent of the PNM stage. This study demonstrated the usefulness of the Echinococcus Western Blot IgG assay as an additional serological test for the follow-up of patients with alveolar echinococcosis.

Possible anaphylactic reaction due to pulmonary hydatid cyst rupture following blunt chest trauma: a case report and review of the literature.

A hydatid cyst is a parasitic disease caused by the tapeworm Echinococcus granulosus. It is endemic in many areas, including New Zealand, Australia, and the Mediterranean region. Pulmonary hydatid disease can be diagnosed incidentally in asymptomatic patients or may cause symptoms such as cough, chest pain, dyspnea, fever, and hemoptysis both in patients with ruptured and nonruptured cysts. Anaphylactic reaction is a rare presentation of pulmonary hydatid cyst disease. In this case report, we report an unusual anaphylactic reaction following pulmonary hydatid cyst rupture secondary to blunt chest trauma.

[Parasite-host relationships and treatment]. / Le parasite et ses relations avec ses hôtes.

Alveolar echinococcosis (AE) is a parasitic disease caused by intrahepatic growth of the larval stage of the cestode Echinococcus multilocularis. The main definitive host in Europe is the fox. The adult worms live in the fox intestine and their oncospheres are disseminated by faeces. Wolves, dogs and cats may also serve as definitive hosts. Small rodents--especially voles in Europe and small lagomorphs in Asia--are the natural intermediate hosts. The tumour-like larva is composed of multiple vesicles which produce protoscoleces, the fertile stage of the E. multilocularis metacestode. Carnivores are infected by preying on infected rodents. Like rodents, humans are intermediate hosts and are infected either by eating uncooked vegetables and berries contaminated by faeces of infected carnivores, or by touching such animals. Humans are naturally resistant to metacestode development. Genetic characteristics are involved in susceptibility/resistance to E. multilocularis metacestodes. In humans and other intermediate animal hosts, immune suppression enhances parasite growth, which is normally controlled by cytotoxic mechanisms and delayed-type hypersensitivity. Tolerance of E. multilocularis is due in part to parasite characteristics (especially carbohydrate antigens of the laminated layer) and in part to the "anti-inflammatory/tolerogenic" cytokines IL-10 and TGF-beta. Treatment with interferon-a restores a cytokine balance favorable to the host and might be a new therapeutic option for AE patients. Vaccination is a scientifically sound but economically and politically Utopian means of preventing the disease. Prevention thus relies on simple lifestyle measures: cooking potentially contaminated food, regular treatment of domestic animals with praziquantel, and precautions when touching potentially infected definitive hosts (foxes and dogs).

Immunomodulative effect of glucan and/or glucan supplemented with zinc in albendazole therapy for murine alveolar echinococcosis.

The effect of glucan immunomodulator (GI) and glucan supplemented with zinc (GIZn) administered separately or with albendazole (ABZ) on cellular immunity of mice with alveolar echinococcosis was observed. The stimulative effect of GI and GI + ABZ therapy on proliferative response of T lymphocytes was prolonged by GIZn or GIZn + ABZ from week 6 to 14 postinfection (p.i.). The increased proliferation of B lymphocytes was observed during combined therapies GI + ABZ and GIZn + ABZ from week 6 to 12 p.i. Number of splenic CD4 T cells in mice with GI or GI + ABZ therapy was increased only on weeks 6 and 8 p.i. GIZn and GIZn + ABZ therapy prolonged this stimulation from week 6 to 14 p.i. Serum concentration of interferon-gamma (IFN-gamma) was increased after GIZn therapy and reduced after GI therapy from week 8 to 12 p.i. GIZn + ABZ therapy had the highest effect on the IFN-gamma rise from week 8 to 22 p.i. Both GI and GIZn inhibited the serum concentration of interleukin-5 (IL-5) from week 6 p.i. The production of superoxide anion was increased after GI therapy from week 6 to 14 p.i. and after GI + ABZ or GIZn + ABZ therapies from week 12 to 18 p.i. The most effective antiparasitic therapy for alveolar echinococcosis was reached by GIZn + ABZ therapy.

A correlative study of ultrasound with serology in an area in China co-endemic for human alveolar and cystic echinococcosis.

We correlated ultrasound (US) imaging classifications for human alveolar echinococcosis (AE) and cystic echinococcosis (CE) with serology (ELISA and immunoblotting (IB) incorporating native and recombinant/purified echinococcal antigens) in community surveys (2001-2003) and follow-up (2002 and 2003) of US-confirmed cases in Ningxia, China. One hundred and seventy-one cases (96 with AE, 75 with CE) were identified; of these, US classification and serological data were obtained for 142 and 112 cases, respectively. Seropositive-rates increased in CE patients with highly viable unilocular cyst lesions (Types CL, CE 1 or CE 2) to degenerating primary lesions (CE 3), but then decreased in subjects with inactive (CE 4) or dead (CE 5) cysts. In contrast, there was a constant increase in seropositivity from the early (P1, P2) to the advanced stages (P3, P4) with AE cases. For US-confirmed cases, follow-up by US combined with serology is invaluable for studying the clinical progression of echinococcosis and for detecting recurrent cysts or reinfection post-treatment.

The immunodiagnosis of Echinococcus multilocularis infection.

Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of Echinococcus multilocularis. The infection can have fatal consequences in humans if treatment is not provided, so early diagnosis is fundamental for initiating treatment and reducing morbidity and mortality. In addition, detection of the parasite in the definitive host plays a central role in epidemiological studies and surveillance programmes for control of AE. This review presents an overview of the present situation regarding the immunodiagnosis of E. multilocularis infection. Special attention is given to the description of the native, partially purified and recombinant antigens available currently for immunodiagnostic purposes. Recent advances in the primary serodiagnosis and follow-up of AE patients are highlighted, including the detection of specific cytokine profiles. Progress in the immunodiagnosis of intestinal E. multilocularis infection in definitive hosts, particularly the detection of excretory-secretory and integument products of the worm in faeces (copro-antigens) by ELISA, is also discussed.

Echinococcus multilocularis metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococcosis patients.

Infection with the cestode Echinococcus multilocularis causes human alveolar echinococcosis (AE), a life-threatening disease affecting primarily the liver. Despite the severity of AE, clinical symptoms often develop only many years after infection, which suggests that E. multilocularis has developed mechanisms which depress anti-parasite immune response, thus favouring immune evasion. In this study we examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable E. multilocularis vesicles and E. multilocularis vesicle fluid antigen in vitro. After 48 h of co-culture, E. multilocularis metacestode culture supernatant and E. multilocularis vesicles depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-alpha and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-alpha was reduced in AE patients, which was accompanied by an increased number of CD4+ CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to E. multilocularis metacestode in AE patients. Instead the production of interferon (IFN)-gamma and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both regulatory and inflammatory Th1 and Th2 cytokines and chemokines. Such a mixed profile might be required for limiting parasite growth but also for reducing periparasitic tissue and organ damage in the host.

Characterization of various recombinant antigens from Echinococcus multilocularis for use in the immunodiagnosis.

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser23-Glu176 of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen BI did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.