Bisphenol A induces protection through modulation of the immune response against the helminth parasite Taenia crassiceps.

AIMS: Industrial growth has increased the exposure to endocrine disruptor compounds (EDCs) in all organisms. Bisphenol A (BPA), an EDC, has been demonstrated to be involved in the susceptibility to parasite infections. However, few studies have analysed this connection in more depth. The aim of this study was to determine whether early BPA exposure in female mice affects the systemic immune response and the susceptibility to Taenia crassiceps infection. METHODS AND RESULTS: BALB/c mice were exposed to BPA at post-natal day 3. At 6 weeks of age, they were inoculated with T crassiceps larvae and, 2 weeks later, were euthanized. The number of parasites was quantified. By flow cytometry, in the spleen, the peripheral and mesenteric lymph nodes, the different innate and adaptive immune cell modulation was analysed, and RT-PCR cytokine expression was also evaluated. BPA induced a reduction of 40% in parasite load. BPA treatment modulated some lineages of the innate immune response and caused slight changes in cells belonging to the adaptive immune response. Additionally, BPA enhanced the type 2 cytokine profile. CONCLUSION: Neonatal BPA treatment in female mice affects not only the percentage of different immune cells but also their ex vivo cytokine gene expression, decreasing T crassiceps cysticercosis susceptibility.

Seroepidemiological evidence for Taenia solium taeniasis/cysticercosis in three Venezuelan rural communities.

Taenia solium is the most common parasite infection of the brain, causing neurocysticercosis and typically found in rural communities with free-ranging pigs. Identification of transmission in rural areas is essential for its control. Risk factors and transmission of the parasite were evaluated in three rural Venezuelan communities (Valle del Rio and Potrero Largo, Cojedes state; and Palmarito, Portuguesa state) by a questionnaire (112 households) and coprological (492 samples) and serological (433 human and 230 porcine sera) analysis, respectively. Typical risk factors were found in all three communities: free-foraging pig husbandry, deficient sanitary conditions, high open defecation and ignorance of the parasite life cycle. Coprological examinations revealed a high level of soil-transmitted parasites. Importantly, two T. solium adult worm carriers were identified in each of the three communities. Anti-metacestode antibodies and the HP10 secreted metacestode glycoprotein were detected at significant levels in human and porcine sera in Valle del Rio, Potrero Largo and Palmarito. In conclusion, these communities may be considered to be endemic for taeniasis/cysticercosis, and the instigation of an appropriate control programme is recommended.

Parasites and epilepsy: Understanding the determinants of epileptogenesis.

There is a large body of evidence suggesting that parasites could be a major preventable risk factor for epilepsy in low- and middle-income countries. We review potentially important substrates for epileptogenesis in parasitic diseases. Taenia solium is the most widely known parasite associated with epilepsy, and the risk seems determined mainly by the extent of cortical involvement and the evolution of the primary cortical lesion to gliosis or to a calcified granuloma. For most parasites, however, epileptogenesis is more complex, and other favorable host genetic factors and parasite-specific characteristics may be critical. In situations where cortical involvement by the parasite is either absent or minimal, parasite-induced epileptogenesis through an autoimmune process seems plausible. Further research to identify important markers of epileptogenesis in parasitic diseases will have huge implications for the development of trials to halt or delay onset of epilepsy.

Anti-GK1 antibodies damage Taenia crassiceps cysticerci through complement activation.

Taeniasis-cysticercosis, a zoonosis caused by Taenia solium, is prevalent in underdeveloped countries, where marginalization promotes its continued transmission. Pig cysticercosis, an essential stage for transmission, is preventable by vaccination. An efficient multiepitope vaccine against pig cysticercosis, S3Pvac, was developed. Previous studies showed that antibodies against one of the S3Pvac components, GK-1, are capable of damaging T. solium cysticerci, inhibiting their ability to transform into the adult stage in golden hamster gut. This study is aimed to evaluate one of the mechanisms that could mediate anti-GK-1 antibody-dependent protection. To this end, pig anti-GK-1 antibodies were produced and purified by using protein A. Proteomic analysis showed that the induced antibodies recognized the respective native cysticercal protein KE7 (Bobes et al. Infect Immun 85:e00395-17, 2017) and two additional T. solium proteins (endophilin B1 and Gp50). A new procedure to evaluate cysticercus viability, based on quantifying the cytochrome c released after parasite damage, was developed. Taenia crassiceps cysticerci were cultured in the presence of differing amounts of anti-GK-1 antibody and complement in a saturating concentration, along with the respective controls. Cysticercus viability was assessed by recording parasite motility, trypan blue exclusion, and cytochrome c levels in cysticercal soluble extract. Anti-GK-1 antibody significantly increased cysticercus damage as measured by all three methods. Parasite evaluation by electron microscopy after treatment with anti-GK-1 antibody plus complement demonstrated cysticercus damage as shorter, capsule-severed microtrichia; a decrease in glycocalyx length with respect to untreated cysts; and disaggregated desmosomes. These results demonstrate that anti-GK-1 antibodies damage cysticerci through classic complement activation.

Co-infection: the outcome of Plasmodium infection differs according to the time of pre-existing helminth infection.

Although helminth-Plasmodium coinfections are common in tropical regions, the implications of this co-existence for the host immune response are poorly understood. In order to understand the effect of helminth infection at different times of coinfection on the immune response against Plasmodium infection, BALB/c mice were intraperitoneally infected with Taenia crassiceps (Tc). At 2 (Tc2) or 8 (Tc8) weeks post-infection, mice were intravenously infected with 1 × 103 Plasmodium yoelii (Py) 17XL-parasitized red blood cells. Py 17XL-single-infected mice developed cachexia, splenomegaly, and anemia, and died at 11 days post-infection. Importantly, Tc2 + Py-coinfected mice showed increased survival of 58% on day 11, but developed pathology (cachexia and splenomegaly) and succumbed on day 18 post-coinfection, this latter associated with high levels of IL-1ß and IL-12, and reduced IFN-γ in serum compared with Py 17XL-single-infected mice. Interestingly, Tc8 + Py-coinfected mice showed increased survival up to 80% on day 11 and succumbed on day 30 post-coinfection. This increased survival rate conferred by chronic helminth infection was associated with a decreased pathology and mixed inflammatory-type 1/anti-inflammatory-type 2 immune profile as evidenced by the production of high levels of IL-12 and IL-10, and reduced TNF-α from macrophages, high levels of IL-4 and IL-10, and low levels of IFN-γ from spleen cells. Also high serum levels of IL-1ß, TNF-α, IL-12, IL-4, and IL-10, but a significant reduction of IFN-γ were observed. Together, these data indicate that polarization of the cell-mediated response modulated by a pre-existing helminth infection differentially impacts on the host immune response to Py 17XL in a time-dependent manner.

Recent advancements and new perspectives in animal models for Neurocysticercosis immunopathogenesis.

Neurocysticercosis (NCC), one of the most common parasitic diseases of the central nervous system, is caused by Taenia solium. This parasite involves two hosts, intermediate hosts (pig and human) and a definitive host (human) and has various stages in its complex life cycle (eggs, oncosphere, cysticerci and adult tapeworm). Hence, developing an animal model for T. solium that mimics its natural course of infection is quite challenging. We have reviewed here the animal models frequently used to study immunopathogenesis of cysticercosis and also discussed their usefulness for NCC studies. We found that researchers have used mice, rats, guinea pigs, dogs, cats and pigs as models for this disease with varying degrees of success. Mice and rats models have been utilized extensively for immunopathogenesis studies due to their relative ease of handling and abundance of commercially available reagents to study these small animal models. These models have provided some very exciting results for in-depth understanding of the disease. Of late, the experimentally/naturally infected swine model is turning out to be the best animal model as the disease progression closely resembles human infection in pigs. However, handling large experimental animals has its own challenges and limitations.

[(CHANGE IN THE DIAGNOSTIC CHARACTERISTICS OF ANTIBODIES TO CYSTICERCUS CELL ULOSAE IN THE SERUM SAMPLE COLLECTION IN RELATION TO THE PERIOD OF STORAGE DURING DEEP FREEZING)].

The paper gives the results of experimental studies determining the preservation of antibodies to C.cellulosae in the serum in relation to the period of their storage during deep freezing. These studies, as applied to parasitic pathology, have been conducted for the first time and are of practical medical value in determining optimal procedures and periods of serum storage without a loss of their diagnostic characteristics.

Anamnestic responses in pigs to the Taenia solium TSOL18 vaccine and implications for control strategies.

Specific antibody responses were assessed in pigs immunized with the Taenia solium vaccine TSOL18. Anti-TSOL18 responses were compared 2 weeks after secondary immunization, where the interval between primary and secondary immunization was 4, 8, 12, 16 or 20 weeks. All animals responded to the vaccine and there was no diminution in antibody responses in animals receiving their second injection after an interval up to 20 weeks. Pigs receiving vaccinations at an interval of 12 weeks developed significantly increased antibody responses compared with animals receiving immunizations 4 weeks apart (P = 0.046). The ability to deliver TSOL18 vaccination effectively where the revaccination schedule can be delayed for up to 12-16 weeks in pigs increases the options available for designing T. solium control interventions that incorporate TSOL18 vaccination.

Behavioral and hormonal changes associated with the infective dose in experimental taeniasis in golden hamsters (Mesocricetus auratus).

INTRODUCTION: It has been reported that behavioral changes relate to infection in different parasitoses. However, the relation between the extent of the behavioral changes and the magnitude of the infection has been scarcely studied. The aim of this study was to evaluate the relationship between different doses of infection and the behavioral changes induced in the experimental Taenia pisiformis taeniasis in golden hamsters. METHODS: Groups of nine hamsters were infected with three or six T. pisiformis metacestodes. The locomotor activity was quantified daily in an open field test during the 21 days after infection; anxiety test was performed in an elevated plus-maze with a dark/light area at 7, 14 and 21 days post-infection, and serum cortisol levels were determined by radioimmunoassay before infection and at day 22 after infection. RESULTS: The challenge itself induced modifications on behavior and cortisol levels in hamsters, with or without successful infection (taenia development). Animals challenged with three metacestodes induced a decrease in locomotor activity and an increase in anxiety in infected animals. A higher and earlier decrease in locomotor activity and increased anxiety levels were observed in hamsters challenged with six cysticerci, which were accompanied by higher levels of sera cortisol at the end of the experiment. At necropsy, 44-55% of hamster became infected with an efficiency of implantation of 22-26%, challenged with three or six cysticerci respectively. CONCLUSION: The challenge of hamsters with metacestodes, promote behavioral changes in an extent dependent on the magnitude of the challenge, disregarding the effectiveness of the infection.

Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis.

The endocrine-immune network during taeniosis by Taenia solium: The role of the pituitary gland.

It is well known that sex hormones play an important role during Taenia solium infection; however, to our knowledge no studies exist concerning the immune response following complete or lobe-specific removal of the pituitary gland during T. solium infection. Thus, the aim of this work was to analyze in hamsters, the effects of lack of pituitary hormones on the duodenal immune response, and their impact on T. solium establishment and development. Thus, in order to achieve this goal, we perform anterior pituitary lobectomy (AL, n = 9), neurointermediate pituitary lobectomy (NIL, n = 9) and total hypophysectomy (HYPOX, n = 8), and related to the gut establishment and growth of T. solium, hematoxylin-eosin staining of duodenal tissue and immunofluorescence of duodenal cytokine expression and compared these results to the control intact (n = 8) and control infected group (n = 8). Our results indicate that 15 days post-infection, HYPOX reduces the number and size of intestinally recovered T. solium adults. Using semiquantitative immunofluorescent laser confocal microscopy, we observed that the mean intensity of duodenal IFN-γ and IL-12 Th1 cytokines was mildly expressed in the infected controls, in contrast with the high level of expression of these cytokines in the NIL infected hamsters. Likewise, the duodenum of HYPOX animals showed an increase in the expression of Th2 cytokines IL-5 and IL-6, when compared to control hamsters. Histological analysis of duodenal mucosa from HYPOX hamsters revealed an exacerbated inflammatory infiltrate located along the lamina propria and related to the presence of the parasite. We conclude that lobe-specific pituitary hormones affect differentially the T. solium development and the gut immune response.

Recombinant protein- and synthetic peptide-based immunoblot test for diagnosis of neurocysticercosis.

One of the most well-characterized tests for diagnosing neurocysticercosis (NCC) is the enzyme-linked immunoelectrotransfer blot (EITB) assay developed at the CDC, which uses lentil lectin-bound glycoproteins (LLGP) extracted from Taenia solium cysticerci. Although the test is very reliable, the purification process for the LLGP antigens has been difficult to transfer to other laboratories because of the need for expensive equipment and technical expertise. To develop a simpler assay, we previously purified and cloned the diagnostic glycoproteins in the LLGP fraction. In this study, we evaluated three representative recombinant or synthetic antigens from the LLGP fraction, individually and in different combinations, using an immunoblot assay (recombinant EITB). Using a panel of 249 confirmed NCC-positive and 401 negative blood serum samples, the sensitivity of the recombinant EITB assay was determined to be 99% and the specificity was 99% for diagnosing NCC. We also tested a panel of 239 confirmed NCC-positive serum samples in Lima, Peru, and found similar results. Overall, our data show that the performance characteristics of the recombinant EITB assay are comparable to those of the LLGP-EITB assay. This new recombinant- and synthetic antigen-based assay is sustainable and can be easily transferred to other laboratories in the United States and throughout the world.

Immunology of Taenia solium taeniasis and human cysticercosis.

The life cycle of Taenia solium, the pork tapeworm, is continuously closed in many rural settings in developing countries when free roaming pigs ingest human stools containing T. solium eggs and develop cysticercosis, and humans ingest pork infected with cystic larvae and develop intestinal taeniasis, or may also accidentally acquire cysticercosis by faecal-oral contamination. Cysticercosis of the human nervous system, neurocysticercosis, is a major cause of seizures and other neurological morbidity in most of the world. The dynamics of exposure, infection and disease as well as the location of parasites result in a complex interaction which involves immune evasion mechanisms and involutive or progressive disease along time. Moreover, existing data are limited by the relative lack of animal models. This manuscript revises the available information on the immunology of human taeniasis and cysticercosis.

A mouse air pouch model for evaluating the immune response to Taenia crassiceps infection.

The experimental system of Taenia crassiceps cysticerci infection in BALB/c mice is considered to be the most representative model of cysticercosis. In our work, mice were sacrificed 7 and 30days after infection, and pouch fluid was collected to determine the number of accumulated cells and the concentrations of IFNγ, IL-2, IL-4, IL-6, IL-10 and nitric oxide. The injection of 50 nonbudding cysticerci into normal mouse dorsal air pouches induced a high level of IFNγ and nitric oxide production relative to the parasite load. The air pouch provides a convenient cavity that allows studying the cellular immunological aspects of the T. crassiceps parasite. The nonbudding cysticerci recovered from the air pouches contained cells that can reconstitute complete cysts in the peritoneal cavity of mice. In conclusion, these results demonstrate that the air pouch model is an alternative tool for the evaluation of the immune characteristics of T. crassiceps infection.

The CD20 homolog Ms4a8a integrates pro- and anti-inflammatory signals in novel M2-like macrophages and is expressed in parasite infection.

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.

Claudin-1, claudin-2 and claudin-11 genes differentially associate with distinct types of anti-inflammatory macrophages in vitro and with parasite- and tumour-elicited macrophages in vivo.

Macrophages altered by various Th2-associated and anti-inflammatory mediators--including IL-4 and IL-13 [inducing alternatively activated macrophages (AAMs)], IL-10 and TGF-ß--were generically termed M2. However, markers that discriminate between AAMs and other M2 remain scarce. We previously described E-cadherin as a marker for AAMs, permitting these macrophages to fuse upon IL-4 stimulation. To identify novel potential contributors to macrophage fusion, we assessed the effect of IL-4 on other adherens and tight junction-associated components. We observed an induction of claudin-1 (Cldn1), Cldn2 and Cldn11 genes by IL-4 in different mouse macrophage populations. Extending our findings to other stimuli revealed Cldn1 as a mainly TGF-ß-induced gene and showed that Cldn11 is predominantly associated with IL-4-induced AAMs. Cldn2 is upregulated by diverse stimuli and is not associated with a specific macrophage activation state in vitro. Interestingly, different claudin genes preferentially associate with M2 from distinct diseases. While Cldn11 is predominantly expressed in AAMs from helminth-infected mice, Cldn1 is the major macrophage claudin during chronic trypanosomiasis and Cldn2 dominates in tumour-associated macrophages. Overall, we identified Cldn1, Cldn2 and Cldn11 as genes that discriminate between diverse types of M2.

Immunological mechanisms involved in the protection against intestinal taeniosis elicited by oral immunization with Taenia solium calreticulin.

Oral immunization with functional recombinant Taenia solium calreticulin (rTsCRT) induces 37% reduction in tapeworm burden in the experimental model of intestinal taeniosis in hamsters. Furthermore, tapeworms recovered from vaccinated animals exhibit diminished length, being frequently found in more posterior parts of the small intestine. The aim of this study was to analyze the immunological mechanisms involved in protection in response to rTsCRT oral immunization. Hamsters were orally immunized with rTsCRT using cholera toxin (CT) as adjuvant, weekly for 4 weeks. Fifteen days after the last boost animals were challenged with four T. solium cysticerci. Reduction in the adult worm recovery and increased transcription of mRNA for IL-4 and IFN-γ in the mucosa of rTsCRT+CT immunized animals were observed. Immunization also induced goblet cell hyperplasia in the mucosa surrounding the implantation site of the parasite. Specific IgG and IgA antibodies in serum and fecal supernatants were detected after the second immunization, being more pronounced after challenge. Our data suggest that oral vaccination with rTsCRT+CT regulates a local expression of IL-4 and IFN-γ, stimulating secretion of IgA that, together with the increase of goblet cells and mucin production, could result in an unfavorable environment for T. solium promoting an impaired tapeworm development.

Taenia crassiceps infection abrogates experimental autoimmune encephalomyelitis.

Helminth infections induce strong immunoregulation that can modulate subsequent pathogenic challenges. Taenia crassiceps causes a chronic infection that induces a Th2-biased response and modulates the host cellular immune response, including reduced lymphoproliferation in response to mitogens, impaired antigen presentation and the recruitment of suppressive alternatively activated macrophages (AAMФ). In this study, we aimed to evaluate the ability of T. crassiceps to reduce the severity of experimental autoimmune encephalomyelitis (EAE). Only 50% of T. crassiceps-infected mice displayed EAE symptoms, which were significantly less severe than uninfected mice. This effect was associated with both decreased MOG-specific splenocyte proliferation and IL-17 production and limited leukocyte infiltration into the spinal cord. Infection with T. crassiceps induced an anti-inflammatory cytokine microenvironment, including decreased TNF-α production and high MOG-specific production of IL-4 and IL-10. While the mRNA expression of TNF-α and iNOS was lower in the brain of T. crassiceps-infected mice with EAE, markers for AAMФ were highly expressed. Furthermore, in these mice, there was reduced entry of CD3(+)Foxp3(-) cells into the brain. The T. crassiceps-induced immune regulation decreased EAE severity by dampening T cell activation, proliferation and migration to the CNS.

Alternatively activated macrophages engage in homotypic and heterotypic interactions through IL-4 and polyamine-induced E-cadherin/catenin complexes.

Alternatively activated macrophages (AAMs), triggered by interleukin-4 (IL-4) and IL-13, play a modulating role during Th2 cytokine-driven pathologies, but their molecular armament remains poorly characterized. Here, we established E-cadherin (Cdh1) as a selective marker for IL-4/IL-13-exposed mouse and human macrophages, which is STAT6-dependently induced during polarized Th2 responses associated with Taenia crassiceps helminth infections or allergic airway inflammation. The IL-4-dependent, arginase-1/ornithine decarboxylase-mediated production of polyamines is important for maximal Cdh1 induction, unveiling a novel mechanism for IL-4-dependent gene transcription. At the macrophage surface, E-cadherin forms a functional complex with the catenins that accumulates at sites of cell contact. Macrophage-specific deletion of the Cdh1 gene illustrates the implication of E-cadherin in IL-4-driven macrophage fusion and heterotypic interactions with CD103(+) and KLRG1(+) T cells. This study identifies the E-cadherin/catenin complex as a discriminative, partly polyamine-regulated feature of IL-4/IL-13-exposed alternatively activated macrophages that contributes to homotypic and heterotypic cellular interactions.