Helminth Products Potently Modulate Experimental Autoimmune Encephalomyelitis by Downregulating Neuroinflammation and Promoting a Suppressive Microenvironment.

A negative correlation between the geographical distribution of autoimmune diseases and helminth infections has been largely associated in the last few years with a possible role for such type of parasites in the regulation of inflammatory diseases, suggesting new pathways for drug development. However, few helminth-derived immunomodulators have been tested in experimental autoimmune encephalomyelitis (EAE), an animal model of the human disease multiple sclerosis (MS). The immunomodulatory activities of Taenia crassiceps excreted/secreted products (TcES) that may suppress EAE development were sought for. Interestingly, it was discovered that TcES was able to suppress EAE development with more potency than dexamethasone; moreover, TcES treatment was still effective even when inoculated at later stages after the onset of EAE. Importantly, the TcES treatment was able to induce a range of Th2-type cytokines, while suppressing Th1 and Th17 responses. Both the polyclonal and the antigen-specific proliferative responses of lymphocytes were also inhibited in EAE-ill mice receiving TcES in association with a potent recruitment of suppressor cell populations. Peritoneal inoculation of TcES was able to direct the normal inflammatory cell traffic to the site of injection, thus modulating CNS infiltration, which may work along with Th2 immune polarization and lymphocyte activation impairment to downregulate EAE development.

A Pipeline for Differential Proteomics in Unsequenced Species.

Shotgun proteomics experiments often take the form of a differential analysis, where two or more samples are compared against each other. The objective is to identify proteins that are either unique to a specific sample or a set of samples (qualitative differential proteomics), or that are significantly differentially expressed in one or more samples (quantitative differential proteomics). However, the success depends on the availability of a reliable protein sequence database for each sample. To perform such an analysis in the absence of a database, we here propose a novel, generic pipeline comprising an adapted spectral similarity score derived from database search algorithms that compares samples at the spectrum level to detect unique spectra. We applied our pipeline to compare two parasitic tapeworms: Taenia solium and Taenia hydatigena, of which only the former poses a threat to humans. Furthermore, because the genome of T. solium recently became available, we were able to prove the effectiveness and reliability of our pipeline a posteriori.

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites.

Taenia solium metacestode preparation in rural areas of sub-Saharan Africa: a source for diagnosis and research on cysticercosis.

BACKGROUND: Taenia solium metacestodes/cysts obtained from pig carcasses constitute a primary source for diagnostic tools used for the detection of human cysticercosis. Data on T. solium cyst preparation in Africa is still scarce but required to establish independent reference laboratories. OBJECTIVES: The aim of the present study is a) to present the likely yield of T. solium cyst material by the use of two different preparation methods in the field and b) to investigate its suitability for immunodiagnosis of human cysticercosis. METHODS: In Zambia, Uganda and Tanzania 670 pigs were screened for T. solium infection. Cysts were prepared by 'shaking method' and 'washing method'. Generated crude antigens were applied in a standard western blot assay. RESULTS: 46 out of 670 pigs (6.9%) were found positive for T. solium (Zambia: 12/367, 3.3%; Uganda: 11/217, 5.1%; Tanzania 23/86, 26.7%). Mean values of 77.7 ml whole cysts, 61.8 ml scolices/membranes and 10.9 ml cyst fluid were obtained per pig. Suitability of collected material for the use as crude antigen and molecular diagnostic techniques was demonstrated. CONCLUSION: This study clearly shows that T. solium cyst preparation in African settings by simple field methods constitutes an effective way to obtain high quality material as source for diagnostic tools and research purposes.

Isolation and identification of lignans from Caulis Bambusae in Taenia with antioxidant properties.

Caulis Bambusae in Taenia is a medicinal preparation from Bambusa tuldoides Munro consisting of skinless slices of the stem (bamboo shavings) and used as a traditional health food in tea, wine, and soup in Asia. Three novel lignans, (-)-7'-epi-lyoniresinol 4,9'-di-O-ß-D-glucopyranoside (7), (-)-lyoniresinol 4,9'-di-O-ß-D-glucopyranoside (8), bambulignan A (10), and seven known lignan compounds (1-6 and 9) were isolated from Caulis Bambusae in Taenia. The structures of the lignans were determined by detailed spectroscopic analysis (HRESIMS, HSQC, HMBC, NOE). All the isolated lignans were tested for antioxidant activities by DPPH and FARP assays. The results showed that the compounds (+)-lyoniresinol 9'-O-ß-D-glucopyranoside (1) and (-)-7'-epi-lyoniresinol 9'-O-ß-D- glucopyranoside (9) have strong free radical scavenging activity and reducing power.

Cloning and characterization of a cathepsin L-like cysteine protease from Taenia pisiformis.

Rabbit cysticercosis, caused by the larval stage of Taenia pisiformis, is a serious parasitic disease of rabbits. It was reported that some cysteine peptidases have potential roles in the pathogenesis of various parasitic infections. To investigate the biochemical characteristics and roles in the pathogenesis/host-invasion of cysteine peptidases, a cDNA sequence encoding for a cathepsin L-like cysteine protease (TpCP) was cloned and identified from the T. pisiformis metacestodes. This sequence was 1220 bp in its length, which included a 1017 bp open reading frame encoding a 339 amino acid peptide. Multiple sequence alignments revealed a 28.9-88.5% similarity with cathepsin L-like cysteine proteases from other helminth parasites and mammals. The recombinant TpCP expressed in Escherichia coli did not show the proteolytic activity by zymography gel assay. However, the TpCP expressed in Pichia pastoris had typical biochemical activities that could hydrolyze rabbit immunoglobulin G, bovine serum albumin and fibronectin. Substrate studies indicated pronounced cleavage of Z-Phe-Arg-AMC. This activity was sensitive to cysteine protease inhibitor E-64 and immunohistochemistry results also indicated that TpCP was distributed as an intense positive reaction in the bladder wall. Our results gave us insights into future studies of TpCP's roles in the infection.

New method to disaggregate and analyze single isolated helminthes cells using flow cytometry: proof of concept.

In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

Pathological, molecular, and biochemical characterization of Coenurus gaigeri in Iranian native goats.

Coenurus gaigeri is the larval stage of Taenia multiceps gaigeri, which infects the muscles of goats and, to a lesser extent, sheep. Metacestodes of the goat such as Coenurus cerebralis, Cysticercus tenuicollis, Cysticercus ovis, and hydatid cysts have been extensively studied. However, because of the uncommon occurrence of C. gaigeri, very few records exist of its biology, pathogenesis, and pathology. Therefore, an investigation was conducted in the summer of 2008 at Shiraz Slaughterhouse, where 2.6% of the goats were infected with intramuscular cysts. Scolices from coenuri were collected for morphologic analysis, and the cysts were identified as C. gaigeri, the intermediate stage of T. multiceps gaigeri. In addition, molecular genetic markers of mitochondrial DNA were applied phylogenetically to resolve the questionable relationship between C. gaigeri and C. cerebralis. All coenuri fluids were aspirated and centrifuged separately; the supernatants were analyzed for biochemical features that included glucose, total protein, urea nitrogen, uric acid, triglycerides, cholesterol, creatinine, calcium, sodium, potassium, alanine aminotransferase, and aspartate aminotransferase. The pathologic changes around the coenuri included mechanical destruction of the affected tissues, associated with degenerative and necrotic changes, infiltration by polymorphonuclear and mononuclear cells, proliferation of fibroblasts, and development of adventitious tissue. Results based on phylogenetic analysis of the mitochondrial DNA (CO1 and ND1) suggest that the larval stages of T. multiceps gaigeri and C. cerebralis, which showed similar morphological criteria, are monophyletic species. However, C. gaigeri interestingly were situated in the biceps femoris, triceps, and abdominal muscles without localization in the nervous system.

A descriptive study of the occurrence and significance of lipids in Taenia hydatigena eggs.

The aim of this work was to investigate the lipid content of Taenia hydatigena eggs and to evaluate the role of lipids in the maintenance of embryo viability. The total lipid content of the egg was 4.5% (w/w). Five classes of neutral lipids were identified: esterified cholesterol, free cholesterol, triacylglycerols, diacylglycerols and free fatty acids. Our results suggest that triacylglycerols play a key role in the maintenance of embryo viability. In addition, we found that T. hydatigena eggs remain metabolically active by mobilisation of stored triacylglycerols. This study contributes to the understanding the survival strategies of a member of the Taeniidae family in the environment outside the host.

Localisation of three host-protective oncospheral antigens of Taenia ovis.

Immunohistochemistry, confocal immunofluorescence and immunogold labelling were used to determine the localisation of the host-protective antigens To16, To18 and To45W in Taenia ovis oncospheres. During maturation of the adult tapeworm the antigens were initially seen as diffuse staining in the developing oncospheres but in mature oncospheres four distinct cells stained positively for the antigens. Confocal fluorescence microscopy using different fluorophores revealed that each of the antigens co-localises within the same cells in the oncosphere. No surface localisation was seen in non-activated or recently activated parasites. Immunogold labelling of non-activated oncosphere sections viewed in transmission electron microscopy revealed labelling of bilateral cells, however the identities of these cells was unclear due to deficiencies in the current level of understanding of oncosphere ultrastructure. Localisation of all the antigens changed dramatically after oncospheres were activated in vitro with each of the antigens being dispersed more generally throughout the parasite parenchyma. During development of the parasites in in vitro culture, surface localisation of the proteins was seen in parasites after 3 or more days in culture. All three antigens were found to be completely absent in parasites by 15 days of culture. The location of the host-protective antigens suggests that initially the invading oncospheres are not susceptible to vaccine-induced antibody and complement mediated attack, but that as the parasites mature, the host-protective antigens come to be associated with the parasite's surface, rendering them susceptible to immune attack.

Immunoblot patterns of Taenia asiatica taeniasis.

Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97 kDa and 21.5 kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and 21.5 kDa against T. asiatica infected sera. Antigens of adult Taenia saginata revealed 110-100, 66, 58-56, and 46 kDa immunoblot bands against T. asiatica infected sera. Antigens of adult Taenia solium also revealed 99-97, 68-66, and 46 kDa bands against T. asiatica infected sera. The immunoblot band of 21.5 kDa exhibited specificity to T. asiatica.

Identification, expression, characterization, and immunolocalization of lactate dehydrogenase from Taenia asiatica.

From the expression sequence tags (ESTs) of Taenia asiatica, an EST containing a putative open reading frame of 993 bp was identified as lactate dehydrogenase (TaLDH) homologue. Recombinant TaLDH (rTaLDH) was expressed in Escherichia coli BL21/DE3 and purified. rTaLDH had a specific LDH activity and could be recognized in serum from swine or patient infected with T. asiatica. TaLDH was immunolocalized on the tegument of T. asiatica adult and embryonic membrane of oncosphere. Our study suggested that TaLDH is a potential drug target and candidate antigen for immunodiagnosis and vaccine for taeniasis and viscero-cysticercosis caused by T. asiatica.

Immunoblot Patterns of Taenia asiatica Taeniasis

Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97 kDa and 21.5 kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and 21.5 kDa against T. asiatica infected sera. Antigens of adult Taenia saginata revealed 110-100, 66, 58-56, and 46 kDa immunoblot bands against T. asiatica infected sera. Antigens of adult Taenia solium also revealed 99-97, 68-66, and 46 kDa bands against T. asiatica infected sera. The immunoblot band of 21.5 kDa exhibited specificity to T. asiatica.

Taenia crassiceps organic acids detected in cysticerci.

Taenia crassiceps cysticerci is used as an experimental model to cysticercosis studies; however there are subcutaneous cases of cysticercosis caused by these cysticerci. It remains unclear in the literature the energetic and fatty acid metabolism in cestodes. Its metabolic study may provide knowledge of pathways that may serve as potential anti-helminthic drugs sites of action. In this work we studied the citric acid cycle organic acids and the fatty acid oxidation in cysticerci removed from mice with 21 and 42 days of infection in two different evolutive stages: growing and final. The organic acids were extracted using perchloric acid and analyzed by HPLC methodology. We found significant statistically differences in oxalate, malate, lactate, and beta-hydroxybutirate concentrations between cysticerci. These results indicate the aerobic metabolism in vivo in spite of the low oxygen concentration of its habitat, and also indicate the presence of fatty acid oxidation as an alternative energetic source.

Carbohydrate components of Taenia crassiceps metacestodes display Th2-adjuvant and anti-inflammatory properties when co-injected with bystander antigen.

Common helminth infections promote Th2-skewed immune responses in their hosts. We have studied the role of intact carbohydrate structures on Taenia crassiceps compounds in the induction of biased type 2 and anti-inflammatory immune responses on peptide-stimulated T cells by using DO11.10 transgenic (OVA Tg) mice. While OVA Tg mice co-injected with OVA peptide (323-339) (OVA(323-339)) plus intact Taenia soluble antigens (iTSA) displayed significantly higher titers of OVA-specific IgG1 and total IgE, low amounts of these antibodies were detectable in sera from OVA Tg mice co-injected with OVA(323-339) plus periodate-carbohydrate altered TSA (paTSA). Spleen cells from OVA Tg mice failed to efficiently produce OVA-specific IFN-gamma but displayed higher IL-4, IL-5 and IL-10 production when they received OVA(323-339) plus iTSA, compared with OVA Tg mice similarly co-injected with OVA(323-339) plus paTSA. Moreover, after in vivo stimulation with OVA(323-339) plus iTSA, spleen cells did show elevated mRNA transcripts for Arginase 1, Ym1, IL-4, IL-10, TGF-beta, and Mannose Receptor (MR) genes, all them associated with Th2-type and anti-inflammatory responses. Similar results were obtained using TLR4 mutant mice. Together these findings suggest that carbohydrate components in TSA are involved in modulating immune responses to bystander antigens and that do not signal via TLR4.

Cadmium and lead concentrations in Skrjabinotaenia lobata (Cestoda: Catenotaeniidae) and in its host, Apodemus sylvaticus (Rodentia: Muridae) in the urban dumping site of Garraf (Spain).

The present study evaluates the parasitological model constituted by the wood mouse (Apodemus sylvaticus) and its intestinal cestode (Skrjabinotaenia lobata) as a potential bioindicator of Cd and Pb in the urban dumping site of Garraf near the city of Barcelona (Spain) and in Begues (reference site). Tissues and respective S. lobata specimens of 38 wood mice captured in Garraf and Begues were analyzed for Cd and Pb by means of ICP-MS. Higher cadmium levels in S. lobata were found only in respect to the muscular levels of their hosts. Nevertheless, lead levels were 8.5-, 53.2- and 81.4-fold higher in S. lobata than kidney, liver and muscle levels of A. sylvaticus from Garraf, respectively. Thus, the proposed model seems to be a promising bioindicator to evaluate environmental lead exposure in terrestrial habitats. In addition, all available data on lead bioaccumulation by cestode parasites of terrestrial mammals are generally discussed.

[Epidemiological investigation of Taenia saginata asiatica in Duyun, Guizhou and detection of amino acids and elements of adult worms].

OBJECTIVE: To study epidemiological factors of taeniasis and to detect amino acid and element components of adult worms in Duyun of Guizhou Province. METHODS: 1. Traditional methods were used for epidemiological investigation. 2. Automatic amino acid analyzer and bioassay were applied for the detection. RESULTS: Among 70 persons with clinical symptoms, 25 patients (24 men and 1 woman) were found to have adult taenia worms in their faeces after taking Areca catechu L. and other drugs. Sixteen amino acids and 12 elements were determined in adult worms. CONCLUSION: Duyun area in Guizhou is a highly endemic area of taeniasis. The pathogenic parasite is identified as Taenia saginata asiatica. Its clinical symptoms are similar to that of Taenia saginata saginata.

PCR tools for the differential diagnosis of Taenia saginata and Taenia solium taeniasis/cysticercosis from different geographical locations.

The potential value of PCRs in the species-specific diagnosis of have been investigated, using samples of T. saginata and T. solium from different geographical areas. The PCRs examining inter-species differences were based on the sequence of the HDP2 DNA fragment, specific for T. saginata/T. solium, and the sequence of the rDNA internal transcribed spacer 1 and spacer 2 (ITS-1 and ITS-2). This PCR analysis of DNA isolates confirmed morphologic diagnosis and allowed the speciation of samples too small or fragmented for morphologic identification, with clear and consistent inter-species differences between T. saginata (twenty-two) and T. solium (three) geographical isolates. Possible intra-species genomic variability, within these species, was similarly studied through analysis of PCR amplification products (PCR-RFLP) and only encountered one exceptional T. saginata isolate from Kenya, which yielded a unique PCR-RFLP pattern, different from T. saginata DNA of Mexican (one sample) and Spanish (seven samples) origin.

Structure and antigenicity of the major glycolipid from Taenia solium cysticerci.

Lipids were extracted from cysticerci of the human tapeworm Taenia solium isolated from various infected pigs and analysed by two-dimensional thin-layer chromatography. These consisted of both alkali-labile and alkali-stable glycolipids, and phosphorylated non-glycosylated lipids. Because abundant and immunogenic glycolipids of parasites have been implicated in host-parasite interactions, the major lipid, an alkali-stable glycolipid, was purified by chromatography and its structure and antigenicity were determined. The structure of the major glycolipid of T. solium, GSL-I, was elucidated through a combination of chemical degradative methods, gas chromatography/mass spectrometry analyses of the degradative products, matrix-assisted-laser desorption/ionisation time of flight mass spectrometry and nuclear magnetic resonance spectroscopy. This analytical strategy led to the identification of a family of beta-galactosylceramides composed mainly of phytosphinganine (2-hydroxylated sphinganine) N-acylated by C16-C24 fatty acids, with the predominance of 2-hydroxylated homologues. Enzyme-linked immunosorbent assay showed no correlation between the antibody titres directed against GSL-I in the human sera and the infective status; in contrast, a very high specific immunoreactivity and a sensitivity above 50% were observed when GSL-I was tested with cerebrospinal fluids from well characterised infected humans. Thus, although these results do not support the use of GSL-I alone as an antigen for the detection of neurocysticercosis, its use as part of an antigen cocktail for the diagnosis of the disease in cerebrospinal fluids merits further investigations.