Schistosoma haematobium total antigen induces increased proliferation, migration and invasion, and decreases apoptosis of normal epithelial cells

Schistosome worms are blood-dwelling flukes that cause chronic infection in more than 200 million people and are thought to be responsible for 500,000 deaths annually. During infection with Schistosoma haematobium, eggs are deposited in the mucosa and submucosa of the bladder and lower ureters. Squamous cell carcinoma (SCC) of the bladder is a long-term sequela of chronic infection. The mechanisms underlying the association between S. haematobium and SCC of the bladder are largely unknown, with all reports to date exclusively demonstrating epidemiological evidence linking S. haematobium infection with SCC of the bladder. We hypothesised that the parasite antigens might induce alterations in epithelial cells towards cancer. For this we used Chinese Hamster Ovary (CHO) cells and treated the cells in culture with S. haematobium total antigen (Sh). Our results showed increased proliferation, increased S-phase and decreased apoptosis, as well as down-regulation of tumour suppressor p27 and up-regulation of anti-apoptotic molecule Bcl-2 in Sh-treated cells compared with controls. We also found increased migration and invasion. To our knowledge, this is the first report demonstrating alterations of normal epithelial cells as a direct effect of S. haematobium antigens.     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

Metabolic alterations in the hamster co-infected with Schistosoma japonicum and Necator americanus

Co-infection with hookworm and schistosomes is a common phenomenon in sub-Saharan Africa, as well as in parts of South America and southeast Asia. As a first step towards understanding the metabolic response of a hookworm-schistosome co-infection in humans, we investigated the metabolic consequences of co-infection in an animal model, using a nuclear magnetic resonance (NMR)-based metabolic profiling technique, combined with multivariate statistical analysis. Urine and serum samples were obtained from hamsters experimentally infected with 250 Necator americanus infective L₃ and 100 Schistosoma japonicum cercariae simultaneously. In the co-infection model, similar worm burdens were observed as reported for single infection models, whereas metabolic profiles of co-infection represented a combination of the altered metabolite profiles induced by single infections with these two parasites. Consistent differences in metabolic profiles between the co-infected and non-infected control hamsters were observed from 4 weeks p.i. onwards. The predominant metabolic alterations in co-infected hamsters consisted of depletion of amino acids, tricarboxylic acid cycle intermediates (e.g. citrate and succinate) and glucose. Moreover, alterations of a series of gut microbial-related metabolites, such as decreased levels of hippurate, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid and trimethylamine-N-oxide, and increased concentrations of 4-cresol glucuronide and phenylacetylglycine were associated with co-infection. Our results provide a first step towards understanding the metabolic response of an animal host to multiple parasitic infections.     

Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina

Extracts of Prunella vulgaris have been shown to exert antiestrogenic effects. To identify the compounds responsible for these actions, we isolated the constituents of P. vulgaris and tested their individual antiestrogenic effects. Rosmarinic acid, caffeic acid, ursolic acid (UA), oleanolic acid, hyperoside, rutin and betulinic acid (BA) were isolated from the flower stalks of P. vulgaris var. lilacina Nakai (Labiatae). Among these constituents, UA and BA showed significant antiestrogenic effects, measured as a decrease in the mRNA level of GREB1, an estrogen-responsive protein; the effects of BA were stronger than those of UA. UA and BA were capable of suppressing estrogen response element (ERE)-dependent luciferase activity and expression of estrogen-responsive genes in response to exposure to estradiol, further supporting the suppressive role of these compounds in estrogen-induced signaling. However, neither UA nor BA was capable of suppressing estrogen signaling in cells ectopically overexpressing estrogen receptor α (ERα). Furthermore, both mRNA and protein levels of ERα were reduced by treatment with UA or BA, suggesting that UA and BA inhibit estrogen signaling by suppressing the expression of ERα. Interestingly, both compounds enhanced prostate-specific antigen promoter activity. Collectively, these findings demonstrate that UA and BA are responsible for the antiestrogenic effects of P. vulgaris and suggest their potential use as therapeutic agents against estrogen-dependent tumors.     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.     

Schistosoma mansoni: Effect of dietary zinc supplement on egg granuloma in Swiss mice treated with praziqantel

Schistosomiasis is one of the most important parasitic diseases in Egypt and chemotherapy is considered the most effective method of control. This study was conducted to assess the effectiveness of zinc administration against Schistosoma mansoni infection by evaluating the activities of arylesterase and paraoxonase (PON1) enzymes, and the degree of liver damage.One hundred and twenty albino mice were divided into two groups; one was an infected control and the other a treated group which was further subdivided into three according to the praziquantel and zinc supplementation given. Blood and liver samples, collected 10 weeks post-infection, were subjected to parasitological, histopathological, and enzyme assays, and immunological studies. The results showed that dietary zinc supplementation led to marked reduction in worm load, and egg deposition in the liver and intestine.Histopathological examination showed marked reduction in the number and diameter of hepatic granulomas in the treated groups. The activity of arylesterase and PON1 enzymes were partially restored in infected animals receiving zinc. IL-10 mRNA expression was higher in the treated groups than in the infection control group. In conclusion, zinc administration could be a promising adjuvant therapy for S. mansoni infection.     

Histamine signalling in Schistosoma mansoni: Immunolocalisation and characterisation of a new histamine-responsive receptor (SmGPR-2)

In parasitic platyhelminthes, including Schistosoma mansoni, biogenic amines play several important roles in the control of motility, metabolism and reproduction. A bioinformatics analysis of the S. mansoni genome identified approximately 16 full-length G protein-coupled receptors (GPCRs) that share significant homology with aminergic receptors from other species. Six of these sequences are structurally related to SmGPR-1 (formerly SmGPCR), a previously described histamine receptor of S. mansoni, and constitute a new clade of amine-like GPCRs. Here we report the cloning of a second member of this clade, named SmGPR-2. The full-length receptor cDNA was expressed in Saccharomyces cerevisiae and shown to be activated by histamine and 1-methylhistamine, whereas other common biogenic amines had no significant effect. Antagonist assays showed that SmGPR-2 was inhibited by classical biogenic amine antagonists but the pharmacological profile was unlike those of known mammalian histamine receptors. Confocal immunolocalisation studies revealed that SmGPR-2 was expressed in the nervous system and was particularly enriched in the subtegumental neuronal plexus of adult S. mansoni and larvae. The ligand, histamine, was found to be widely distributed, mainly in the peripheral nervous system including the subtegumental plexus where the receptor is also expressed. Finally, SmGPR-2 was shown to be developmentally regulated at the RNA level. Quantitative PCR studies showed it was up-regulated in the parasitic stages compared with cercaria and expressed at the highest level in young schistosomula. The widespread distribution of histamine and the presence of at least two receptors in S. mansoni suggest that this transmitter is an important neuroactive substance in schistosomes.     

Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.     

Plasmodium yoelii: Adverse outcome of non-lethal P. yoelii malaria during co-infection with Schistosoma mansoni in BALB/c mouse model

Plasmodium yoelii and Schistosoma mansoni co-infections were studied in female BALB/c mice aged 4-6 weeks to determine the effect of time and stage of concomitant infections on malaria disease outcome. Patent S. mansoni infection in BALB/c mice increased malaria peak parasitemia and caused death from an otherwise non-lethal, self-resolving P. yoelii malaria infection. Exacerbation of malaria parasitemia occurred during both pre-patent and patent S. mansoni infection resulting in a delay of 4-8 days in malaria parasite resolution in co-infected mice. Praziquantel administered to mice with patent schistosome infection protected from fatal outcome during co-infection. However, this treatment did not completely clear the worm infestation, nor did it reduce the peak malaria parasitemia reached, which was nonetheless resolved completely. Hepatosplenomegaly was more marked in schistosome and malaria co-infected mice compared to either infection separately. The results suggest a complex relationship between schistosome co-infection and malaria disease outcome in which the timing of malaria infection in relation to schistosome acquisition is critical to disease outcome and pathology.     

Identification of new immune induced molecules in the haemolymph of Drosophila melanogaster by 2D-nanoLC MS/MS

Antimicrobial peptides (AMPs) play an important role in the innate immunity of insects. In Drosophila 17 additional immune induced molecules (DIMs) were found in the haemolymph of adult flies upon septic injury. Previous studies using MALDI mass spectrometry combined with Edman degradation, detected AMPs and DIMs of a predominantly large size. By means of 2D-nanoLC ESI MS/MS, 43 DIMs were identified in this study from the haemolymph of Drosophila third instar larvae 12h after challenge with a mixture of Micrococcus luteus and Escherichia coli. Most peptides were derived from known AMP or DIM precursors, but only four peptides were purified and identified before. The majority of the peptides that we detected were smaller in size. Interestingly, two previously unknown peptide precursors were found and hereby related to immune defense. These include CG7738 and CG32185. Many of the identified peptides are post-translationally modified by an N-terminal pyroglutamic acid and/or a C-terminal amide. Haemolymph of control larvae was treated in the same way and revealed only one peptide.     

Schistosoma mansoni: Developmental arrest of miracidia treated with histone deacetylase inhibitors

In the present study, we examined the effect of the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VA) and sodium-butyrate on the metamorphosis of larvae of the human blood-fluke Schistosoma mansoni from the free-swimming miracidia into the intramolluskal sporocyst. We show that HDAC inhibitors block transformation in concentration dependant manner. TSA reversibly blocks this developmental process: only 13 ± 11% of TSA treated miracidia transform into sporocysts in-vitro, compared to 92 ± 3% in the mock-treated control. Other enzyme inhibitors such as cycloheximide or hydroxyurea had no effect on metamorphosis. For treatment of up to 4 h, the effect of TSA was completely reversible. Our data indicates that HDAC activity is necessary for the transformation of S. mansoni miracidia during infection of the snail host.     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.     

Female biased sex-ratio in Schistosoma mansoni after exposure to an allopatric intermediate host strain of Biomphalaria glabrata

For parasites that require multiple hosts to complete their development, the interaction with the intermediate host may have an impact on parasite transmission and development in the definitive host. The human parasite Schistosoma mansoni needs two different hosts to complete its life cycle: the freshwater snail Biomphalaria glabrata (in South America) as intermediate host and a human or rodents as final host. To investigate the influence of the host environment on life history traits in the absence of selection, we performed experimental infections of two B. glabrata strains of different geographic origin with the same clonal population of S. mansoni. One B. glabrata strain is the sympatric host and the other one the allopatric host. We measured prevalence in the snail, the cercarial infectivity, sex-ratio, immunopathology in the final host and microsatellite frequencies of individual larvae in three successive generations.     

Human helminth co-infection: No evidence of common genetic control of hookworm and Schistosoma mansoni infection intensity in a Brazilian community

Strong statistical associations between soil-transmitted helminths and schistosomes are frequently observed in co-endemic human populations, although the underlying explanations remain poorly understood. This study investigates the contribution of host genetics and domestic environment to hookworm and Schistosoma mansoni infection intensity and evaluates the role of genetic and non-genetic factors in co-variation of infection intensity. Detailed genealogical information allowed assignment of 1303 individuals living in the Brazilian community of Americaninhas, Minas Gerais state, to 25 pedigrees (containing between two and 1159 members) residing in 303 households. The prevalence of co-infection with both hookworms and schistosomes was high (38.5%), with significant correlation between Necator americanus and S. mansoni faecal egg counts. Bivariate variance component analysis demonstrated a modest but significant species-specific heritability for intensity of N. americanus (h² = 0.196) and S. mansoni infection (h² = 0.230). However, after accounting for demographic, socio-economic and household risk factors, no evidence for common genetic control of intensity of hookworm and schistosome infection was observed. There was some evidence for residual clustering within households but the majority (63%) of the covariance between N. americanus and S. mansoni infection intensity remained specific to the individual and could not be explained by shared genes, shared environment or other shared demographic, socio-economic or environmental risk factors. Our results emphasize the importance of exposure to hookworm and schistosome infection in driving the association between levels of infection with these species in hosts resident in areas of high transmission and suggest that much of this common exposure occurs outside the home.     

N-Glycosylation patterns of hemolymph glycoproteins from Biomphalaria glabrata strains expressing different susceptibility to Schistosoma mansoni infection

Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (β1-2)-linked xylose or terminal Fuc(α1-3)GalNAc(β1-4)[±Fuc(α1-3)]GlcNAc(β1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host.     

Schistosoma japonicum: A PCR assay for the early detection and evaluation of treatment in a rabbit model

A specific PCR assay for the detection of Schistosoma japonicum DNA in rabbit fecal and serum samples was developed by amplifying a 230-bp fragment from the sequence information of the clone G55A of the highly repetitive retrotransposon SjR2. The minimum amount of DNA detectable using the PCR assay was 0.8 pg, and the expected PCR product was amplified when DNA equivalent of 1.1 egg from feces was used as template. In the meantime, serum anti-worm IgG was examined by ELISA. ELISA gave positive results at 4-6 weeks post-infection depending on the cercarial doses. The parasite eggs were detected in feces at 7 weeks post-infection. In contrast, S. japonicum DNA was detected in sera at first week post-infection, and it became negative at 10 weeks post-treatment, whereas the anti-worm IgG was still at high levels at 23 weeks post-treatment. These data demonstrated that the PCR assay established provides a potential tool for the early diagnosis and therapy evaluation for S. japonicum infection in humans.