Taenia crassiceps-Excreted/Secreted Products Induce a Defined MicroRNA Profile that Modulates Inflammatory Properties of Macrophages.

Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.

Fate of uptaken host proteins in Taenia solium and Taenia crassiceps cysticerci.

During the study of host-parasite relationships in taeniid parasite diseases, including cysticercosis and hydatidosis, reports have described the presence of host proteins in the cyst fluid and tissue of metacestodes. However, the fate or role of host elements inside the parasite remains barely explored. After the publication of genomes of four cestode species, it became clear that these organisms possess a limited biosynthetic capability. The initial goal of the present study was to determine if uptaken host proteins could be a source of essential amino acids for cysticerci. To track the utilization of uptaken proteins, we added metabolically labeled IgG-3H and GFP-3H to the culture medium of Taenia crassiceps cysticerci. Incorporation of labeled amino acid was evaluated by fluorography in cysticerci extracts. Our results showed that the use of uptaken proteins by cysticerci as a source of amino acids appeared negligible. Exploring alternative fates for the host proteins, proteomic analysis of the protein matrix in calcareous corpuscles was carried out. Since T. crassiceps does not contain calcareous corpuscles, proteomic analyses were performed in corpuscles of Taenia solium cysticerci. Our results demonstrated that host proteins represented approximately 70% of protein content in the calcareous corpuscles. The presence of the two major uptaken host proteins, namely albumin and IgG, was also demonstrated by Western blot in the matrix of corpuscles. Our findings strongly suggested that the uptake and disposal of host proteins involve calcareous corpuscles, expanding the physiological role of these mineral concretions to a far more important level than previously proposed.

Hair cortisol and dehydroepiandrosterone concentrations in naturally Taenia solium infected pigs in Tanzania.

The aim of this study was to measure hair cortisol and dehydroepiandrosterone (DHEA) concentrations in naturally Taenia solium infected and non-infected control pigs and assess the effect of an environmental change on the aforementioned parameters. Three hair patches were obtained from 13 T. solium infected and 15 non-infected controls sows, respectively corresponding to 3 time points (prior to, at and approximately two weeks after arrival at the research facility). Cortisol and DHEA were extracted using methanol and analysed by radio immune assay. Mean hair cortisol concentrations were significantly lower (p<0.001) in T. solium infected (4.7±3.0pg/mg) compared to control pigs (9.0±3.7pg/mg) prior to arrival at the research facility, however no significant difference was observed between the two groups at arrival and after approximately two weeks. Similar patterns were also observed for DHEA concentrations (infected pigs 253.9±82.3pg/mg, control pigs 387.7±116.4pg/mg) (p<0.001). Results showed that lean animals had significantly higher cortisol concentrations in both groups, infected and controls pigs, while DHEA was not significantly different between lean and normal animals. Results of this study have shown that an environmental change could have an effect on pigs' hormonal levels suggesting an undergoing adaptation process. After the pigs were kept under the same conditions, fed and watered ad libitum, no significant differences were observed between the groups, but a drop in DHEA concentrations was observed in all the pigs. Weight however had an effect on cortisol levels as lean animals had significantly higher cortisol concentrations in both groups, compared to normal pigs.

Cognitive Performance and Iron Status are Negatively Associated with Hookworm Infection in Cambodian Schoolchildren.

Soil-transmitted helminth (STH) infection has been associated with lower cognitive performance of schoolchildren. To identify pathways through which STH infection might affect school performance, baseline data from a large rice-fortification trial in Cambodian schoolchildren were used to investigate associations between STH infection, micronutrient status, anemia, and cognitive performance. Complete data on anthropometry, cognitive performance, and micronutrient status were available for 1,760 schoolchildren, 6-16 years of age. STH infection was identified using Kato-Katz, whereas cognitive performance was assessed using Raven's Colored Progressive Matrices (RCPM), block design, and picture completion. STH infection was found in 18% of the children; almost exclusively hookwork infection. After adjusting for age and gender, raw cognitive test scores were significantly lower in hookworm-infected children (-0.65; -0.78; -2.03 points for picture completion, RCPM, and block design, respectively; P < 0.05 for all). Hookworm infection was associated with iron status (total body iron), but not with vitamin A and zinc status, nor with inflammation or anthropometry. Body iron was negatively associated with increased intensity of hookworm infection (R = 0.22, P < 0.001). Hookworm infection in Cambodian schoolchildren was associated with lower cognitive performance, an effect most likely mediated through lower body iron. Interventions that are more effective against hookworm infection are needed to contribute to better health and improvement of cognitive performance.

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.

A Taenia crassiceps factor induces apoptosis of spleen CD4+T cells and TFG-ß and Foxp3 gene expression in mice.

This study was undertaken to determine whether a parasite substance produces structural pathology in the mouse spleen. A low-molecular-weight Taenia crassiceps metacestode factor (MF) isolated from the peritoneal fluid of female mice infected with T. crassiceps metacestodes induced pathological and immunological changes in mouse spleen cells in vivo. Electron microscopy and confocal microscopy revealed severe changes in the spleen histoarchitecture of T. crassiceps-infected and MF-treated mice. Apoptotic degenerated spleen cells were observed in the white and red pulps and were more conspicuous in the white pulp of the spleen from the T. crassiceps-infected mice than in that of the MF-treated mice. Flow cytometry analysis revealed that the numbers of spleen CD4+T cells were significantly lower in both experimental groups than in control mice. The ex vivo expression of transforming growth factor (TGF)-ß and factor Foxp3 were significantly higher in splenocytes of the experimental mice than the basal expression observed in the control cells. These findings may have potential applications for a better understanding of the host-parasite relationship in human neurocysticercosis.

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.

Extraintestinal helminth infection reduces the development of colitis-associated tumorigenesis.

Colitis-associated colorectal cancer (CAC) is one of the most common cancers and is closely related to chronic or deregulated inflammation. Helminthic infections can modulate inflammatory responses in some diseases, but their immunomodulatory role during cancer development remains completely unknown. We have analyzed the role of Taenia crassiceps-induced anti-inflammatory response in determining the outcome of CAC. We show that extraintestinal T. crassiceps infection in CAC mice inhibited colonic inflammatory responses and tumor formation and prevented goblet cell loss. There was also increased expression of IL-4 and alternatively activated macrophages markers in colonic tissue and negative immunomodulation of pro-inflammatory cytokine expression. In addition, T. crassiceps infection prevented the upregulation of ß-catenin and CXCR2 expression observed in the CAC mice, which are both markers associated with CAC-tumorigenesis, and reduced the numbers of circulating and colonic CD11b(+)Ly6C(hi)CCR2(+) monocytes. Thus, immunomodulatory activities induced by helminth infections may have a role in the progression of CAC.

Taenia crassiceps infection and its excreted/secreted products inhibit STAT1 activation in response to IFN-γ.

It is well understood that helminth infections modulate the immune responses of their hosts but the mechanisms involved in this modulation are not fully known. Macrophages and dendritic cells appear to be consistently affected during this type of infection and are common target cells for helminth-derived molecules. In this report, we show that macrophages obtained from chronically Taenia crassiceps-infected mice displayed an impaired response to recombinant murine IFN-γ, but not to recombinant murine IL-4, as measured based on the phosphorylation of STAT1 and STAT6, respectively. These macrophages expressed high levels of SOCS3. However, the inhibition of phosphatase activity by orthovanadate restored the IFN-γ response of these macrophages by increasing STAT1 phosphorylation without affecting SOCS3 expression. Therefore, we aimed to identify the phosphatases associated with IFN-γ signaling inhibition and found that macrophages from T. crassiceps-infected mice displayed enhanced SHP-1 expression. Interestingly, the exposure of naïve macrophages to T. crassiceps excreted/secreted products similarly interfered with IFN-γ-induced STAT1 phosphorylation. Moreover, macrophages exposed to T. crassiceps excreted/secreted products expressed high levels of SOCS3 as well as SHP-1. Strikingly, human peripheral blood mononuclear cells that were exposed to T. crassiceps excreted/secreted products in vitro also displayed impaired STAT1 phosphorylation in response to IFN-γ; again, phosphatase inhibition abrogated the T. crassiceps excreted/secreted product-altered IFN-γ signaling. These data demonstrate a new mechanism by which helminth infection and the products derived during this infection target intracellular pathways to block the response to inflammatory cytokines such as IFN-γ in both murine and human cells.

Substance P signaling contributes to granuloma formation in Taenia crassiceps infection, a murine model of cysticercosis.

Cysticercosis is an infection with larval cysts of the cestode Taenia solium. Through pathways that are incompletely understood, dying parasites initiate a granulomatous reaction that, in the brain, causes seizures. Substance P (SP), a neuropeptide involved in pain-transmission, contributes to inflammation and previously was detected in granulomas associated with dead T. crassiceps cysts. To determine if SP contributes to granuloma formation, we measured granuloma-size and levels of IL-1beta, TNF-alpha, and IL-6 within granulomas in T. crassiceps-infected wild type (WT) mice and mice deficient in SP-precursor (SPP) or the SP-receptor (neurokinin 1, NK1). Granuloma volumes of infected SPP- and NK1-knockout mice were reduced by 31 and 36%, respectively, compared to WT mice (P < .05 for both) and produced up to 5-fold less IL-1beta, TNF-alpha, and IL-6 protein. Thus, SP signaling contributes to granuloma development and proinflammatory cytokine production in T. crassiceps infection and suggests a potential role for this mediator in human cystercercosis.

Differential response of antigen presenting cells from susceptible and resistant strains of mice to Taenia crassiceps infection.

Antigen presenting cells (APCs) are critically involved in the interaction between pathogens and the host immune system. Here, we examined two different populations of APCs in mice that are susceptible (BALB/c) or resistant (C57BL/6) to Taenia crassiceps cysticercosis. Bone marrow-derived dendritic cells (BMDCs) from both strains of mice were exposed to T. crassiceps excreted/secreted antigens (TcES) and, at the same time, to the Toll-like receptor (TLR) ligand LPS. BMDCs from BALB/c mice underwent a partial maturation when incubated with TcES and displayed decreased responses to TLR-dependent stimuli associated with low CD80, CD86, CD40 and CCR7 expression and impaired IL-15 production. These BMDCs-induced impaired allogenic responses. In contrast, BMDCs from C57BL/6 mice displayed normal maturation and induced strong allogenic responses. Moreover, the exposure to TcES resulted in a lower production of IL-12 and TNF-alpha by LPS-activated DCs from BALB/c mice compared to C57BL/6 DCs. Three parameters of macrophage activation were assessed during Taenia infection: LPS+IFN-gamma-induced production of IL-12, TNF-alpha and nitric oxide (NO) in vitro; infection-induced markers for alternatively activated macrophages (Arginase-1, RELM-alpha, Ym-1 and TREM-2 expression) and suppressive activity. The maximum response to LPS+IFN-gamma-induced TNF-alpha, IL-12 and NO production by macrophages from both strains of mice occurred 2 wk post-infection. However, as infection progressed, the production of these molecules by BALB/c macrophages declined. While the BALB/c macrophages displayed impaired pro-inflammatory responses, these macrophages showed strong Arginase-1, Ym-1, RELM-alpha and TREM-2 expression. By contrast, C57BL/6 macrophages maintained a pro-inflammatory profile and low transcripts for alternative activation markers. Macrophages from T. crassiceps-infected BALB/c mice showed stronger suppressive activity than those from C57BL/6 mice. These findings suggest that APC activation at both early and late time points during T. crassiceps infection is a possible mechanism that underlies the differential susceptibility to T. crassiceps infection displayed by these mouse strains.

Cytokine response in the intestinal mucosa of hamsters infected with Taenia solium.

Taenia solium grows in experimentally infected hamsters. An inflammatory reaction in the intestinal mucosa surrounding the scolex of the worms is produced. We searched for mRNA of Th1 and Th2 cytokines by in situ hybridization in intestinal biopsies. Hamsters were infected with T. solium cysticerci and necropsied on different days post infection (d.p.i.). Tissue from the small intestine was taken from the area surrounding the tapeworm scolex, fixed, and processed for histology. Antisense probes for the detection of interferon (IFN)-gamma, interleukin (IL)-4, IL-5, and IL-13 were used. Kinetics of each cytokine was defined through detection on specific mRNA by counting the number of positive infected hamsters and of positive cells per 100 enterocytes on different d.p.i. IFN-gamma was detected as of d.p.i. 2; all animals were positive on d.p.i. 4 and 8; and on d.p.i. 16, only 20% were still positive. IL-13 had a pattern similar to IFN-gamma, but all hamsters remained positive until d.p.i. 16 when the experiment was terminated. IL-4 was positive in 40% of infected hamsters on d.p.i. 6. On d.p.i. 8, IL-5 was only detected in 20% but increased to 100% by d.p.i. 16. These data suggest that tapeworms induce a mixed Th1/Th2 response with a polarization toward Th2 at 2 weeks post infection, which may influence the expulsion of worms.

Uptake and secretion of host proteins by Taenia crassiceps metacestodes.

Although the presence of intact host proteins in the cyst fluid of cyclophyllidean metacestodes has been well documented, the underlying reason for protein uptake is poorly understood. To investigate this discrepancy, both the cyst fluid (CF) and excreted/secreted (E/S) proteins were collected in vitro from Taenia crassiceps metacestodes 16 wk postinfection in Balb/cJ female mice. The CF and E/S were subsequently immunoblotted using rabbit anti-mouse whole serum antibodies as a probe. The results show that whole host proteins were not only internalized by metacestodes but also secreted as well. The predominant secreted host protein was 66 kDa and was confirmed to be mouse serum albumin. The amount of secreted albumin decreased daily, whereas the concentration of albumin in the cyst fluid remained consistent. This suggests that the secretion of albumin is a coordinated function rather than a random event. It is probable that albumin cycling may be an evolved mechanism providing multiple benefits for the larvae, including osmoregulation and protection from innate immune responses.

Metabolism of steroid hormones by Taenia solium and Taenia crassiceps cysticerci.

Previous in vitro experiments showed that both, Taenia crassiceps and Taenia solium cysticerci have the ability to metabolize exogenous androstenedione to testosterone. Here we evaluate on the capacity of both cysticerci to synthesize several sex steroid hormones, using different hormonal precursors. Experiments using thin layer chromatography (TLC) showed that both cysticerci were able to produce (3)H-hydroxyprogesterone, (3)H-androstenedione and (3)H-testosterone when (3)H-progesterone was used as the precursor. They also synthesized (3)H-androstenediol and (3)H-testosterone when (3)H-dehydroepiandrosterone was the precursor. In addition, both cysticerci interconverted (3)H-estradiol and (3)H-estrone. These results, strongly suggest the presence and activity of the Delta4 and Delta5 steroid pathway enzymes, 3beta-hydroxysteroid dehydrogenase/Delta(5-4) isomerase-like enzyme (3beta-HSD), that converts androstenediol into testosterone; and the 17beta-hydroxysteroid dehydrogenase that interconverts estradiol and estrone, in both types of cysticerci.

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.

Retarded gastric acid secretion in rats infected with larval Taenia taeniaeformis.

The influence of hepatic larval Taenia taeniaeformis infection on gastric acid secretory activity and gastric mucosal integrity was investigated. After 12 weeks of infection with 2,000 T. taeniaeformis eggs, the gastric pH values of control and infected rats were 4.1+/-0.6 (mean +/- SD) and 8.4+/-0.2, respectively. There was no difference in the basal acid secretion between control (1.7+/-0.7 micro Eq.H(+)/15 min) and infected (1.9+/-0.3) rats. However, infected rats failed to respond to histamine stimulation, the maximum acid output level being 2.8+/-0.4 in the infected rats, compared to 12.9+/-3.3 in control rats. Larval T. taeniaeformis infection resulted in the suppression of gastric acid secretion leading to hypergastrinemia.

Taenia taeniaeformis: increased cell growth and neutral mucus production in the gastric mucosa of the rat with a larval infection.

Gross hyperplasia of the gastric mucosa and excessive mucus production in the stomach occur in rats heavily parasitized with larvae of Taenia taeniaeformis. In this study, a positive correlation between the number of larvae recovered from hepatic cysts and the weight of the stomachs of infected rats was found. By light microscopy, the hyperplasia was restricted to the glandular mucosa. Parietal and chief cells were very rare, and densely PAS-positive mucous cells were the major cell types in the hyperplastic stomach while, in comparison, alcian blue-positive cells were much fewer in number. The isolated gastric mucosa in organ culture had an increased [3H]thymidine incorporation rate in rats infected with T. taeniaeformis. The hexosamine concentration per milligram protein in the hyperplastic stomach mucosa was twice that in the control rat stomach mucosa. By electron microscopy, the apical cytoplasm of the mucous cells was found to be filled with small dark granules. These results indicate that the gastric hyperplasia is caused by stimulation of growth and major differentiation of stem cells to neutral mucus-producing cells.

Absorption studies in patients with parasitic infestation of the small intestine, before and after treatment.

Forty adult patients having intestinal infestation with giardia or with parasitic associations, such as giardia-strongiloides, giardia-taenia solium, were subjected to morphological explorations, iron and vitamin B12 absorption tests, steatorrhea assay and serological tests, before treatment as well as six months and one year after eradication of the infection. On admittance, jejunal morphological lesions were noted only in 15 cases especially in associated infestation, iron depletion in six patients, vitamin B12 malabsorption in five patients and steatorrhea only in two cases. After the lapse of six months and one year, respectively, all the tests ranged within normal values, and the jejunal morphological aspect improved significantly indicating the pathogenetic role of intestinal parasites in the development of selective malabsorption.