Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro.

Cysticercosis is a disease caused by the larval stage of Taenia solium cestodes that belongs to the family Taeniidae that affects a number of hosts including humans. Taeniids tapeworms are hermaphroditic organisms that have reproductive units called proglottids that gradually mature to develop testis and ovaries. Cysticerci, the larval stage of these parasites synthesize steroids. To our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursors to steroid hormones. Therefore, the aim of this paper was to investigate if T. solium tapeworms were able to transform steroid precursors to corticosteroids and sex steroids. T. solium tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were cultured in the presence of tritiated progesterone or androstenedione. At the end of the experiments the culture media were analyzed by thin layer chromatography. The experiments described here showed that small amounts of testosterone were synthesized from (3)H-progesterone by complete or segmented tapeworms whereas the incubation of segmented tapeworms with (3)H-androstenedione, instead of (3)H-progesterone, improved their capacity to synthesize testosterone. In addition, the incubation of the parasites with (3)H-progesterone yielded corticosteroids, mainly deoxicorticosterone (DOC) and 11-deoxicortisol. In summary, the results described here, demonstrate that T. solium tapeworms synthesize corticosteroid and sex steroid like metabolites. The capacity of T. solium tapeworms to synthesize steroid hormones may contribute to the physiological functions of the parasite and also to their interaction with the host.

In vivo albendazole treatment of Taenia crassiceps cysticerci strain WFU: proliferation, damage, and recovery.

Taenia crassiceps has been widely experimented as a model for in vitro and in vivo studies on drug responses. The purpose of this study was to treat BALB/c mice infected with T. crassiceps strain WFU with commercially available albendazole and to analyze the reduction in parasite infrapopulations. Here, we describe the reduction and apparent damage of T. crassicceps WFU cysticerci in infected mice after antihelminthic drug treatment and subsequent inoculation of those treated parasites into a naïve host. We were able to reduce significantly the parasite counts to 33 and 48% after albendazole treatment for 20 or 25 days and compared with the untreated mice. We also observed morphological damage such as the partial blebbing in the tegument and parenchyma of treated parasites, as well as disorganized musculature and the loss of cell membranes in subtegumental tissue section. However, larvae from albendazole-treated mice inoculated into the next host were able to become re-established in the next murine host due, probably, to the survival of proliferative parasite cells.

Steroid synthesis by Taenia crassiceps WFU cysticerci is regulated by enzyme inhibitors.

Cysticerci and tapeworms from Taenia crassiceps WFU, ORF and Taenia solium synthesize sex-steroid hormones in vitro. Corticosteroids increase the 17ß-estradiol synthesis by T. crassiceps cysticerci. T. crassiceps WFU cysticerci synthesize corticosteroids, mainly 11-deoxycorticosterone (DOC). The aim of this work was to investigate whether classical steroidogenic inhibitors modify the capacity of T. crassiceps WFU cysticerci to synthesize corticosteroids and sex steroid hormones. For this purpose, T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, pre-cultured for 24h in DMEM+antibiotics/antimycotics and cultured in the presence of tritiated progesterone ((3)H-P4), androstendione ((3)H-A4), or dehydroepiandrosterone ((3)H-DHEA) plus different doses of the corresponding inhibitors, for different periods. Blanks with the culture media adding the tritiated precursors were simultaneously incubated. At the end of the incubation period, parasites were separated and media extracted with ether. The resulting steroids were separated by thin layer chromatography (TLC). Data were expressed as percent transformation of the tritiated precursors. Results showed that after 2h of exposure of the cysticerci to 100 µM formestane, the (3)H-17ß-estradiol synthesis from tritiated androstenedione was significantly inhibited. The incubation of cysticerci in the presence of (3)H-DHEA and danazol (100 nM) resulted in (3)H-androstenediol accumulation and a significant reduction of the 17ß-estradiol synthesis. The cysticerci (3)H-DOC synthesis was significantly inhibited when the parasites were cultured in the presence of different ketoconazole dosis. The drug treatments did not affect parasite's viability. The results of this study showed that corticosteroid and sex steroid synthesis in T. crassiceps WFU cysticerci can be modified by steroidogenic enzyme inhibitors. As was shown previously by our laboratory and others, parasite survival and development depends on sex steroids, therefore the inhibition of their synthesis is a good starting point exploited in situations where the inhibition of steroidogenesis could help to control the infection for the development of new treatments, or replacement of the usual therapy in resistant parasite infections. We raise the possibility that these drug actions may be beneficially.

Taenia crassiceps WFU cysticerci synthesize corticosteroids in vitro: metyrapone regulates the production.

Taenia solium and Taenia crassiceps WFU cysticerci and tapeworms have the ability to synthesize sex steroid hormones and have a functional 3ß-hydroxisteroid dehydrogenase. Corticosteroids (CS) like corticosterone and dexamethasone have been shown to stimulate in vitro estrogen production by Taenia crassiceps WFU cysticerci. The aim of this work was to study the ability of T. crassiceps WFU cysticerci to synthesize corticosteroids, and the effect of the inhibitor metyrapone on the CS synthesis. For this purpose T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, thoroughly washed and pre-incubated in multiwells for 24 h in DMEM plus antibiotics/antimycotics. The tritiated CS precursor progesterone ((3)H-P4) was added to the culture media and parasites cultured for different periods. Blanks containing the culture media plus the (3)H-P4 were simultaneously incubated. Blanks and parasite culture media were ether extracted and analyzed by thin layer chromatography (TLC) in two different solvent systems. Corticosterone production was measured in the culture media by RIA. In some experiments metyrapone (0.1-0.5 mM) was added for 24, 48 or 72 h. Results showed that cysticerci mainly synthesized tritiated 11-deoxy corticosterone (DOC) and small amounts of corticosterone that was also detected by RIA. Small amounts of (3)H-11-deoxy cortisol were also found. Corticosteroid synthesis was time dependent. The addition of metyrapone significantly inhibited tritiated DOC, deoxycortisol and corticosterone synthesis. These results show for the first time that parasites have the capacity to synthesize CS that is modulated by metyrapone. Data suggest that DOC is the main corticosteroid in the parasites.

The effect of glucocorticoids on sex steroid synthesis in cultured Taenia crassiceps Wake Forest University (WFU) cysticerci.

We have shown previously that cultured Taenia crassiceps Wake Forest University (WFU) and Taenia solium cysticerci, as well as the adult worms, synthesize sex steroid hormones from [3H]steroid precursors and that androgens and oestrogens influence the in vitro development of the parasites. Glucocorticoids (GCs) are used to control the inflammation caused by T. solium cysticerci in the brain. These steroids stimulate oestrogen synthesis in several tissues. Since there is no information on the effect of GC on the endocrine function of cysticerci, we investigated the effect of natural and synthetic GCs on the synthesis of oestrogens in cultured T. crassiceps WFU cysticerci. The cysticerci were obtained from the peritoneal cavity of infected female BALB/c mice; the cysts were washed extensively and pre-cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus antibiotics for 5 days. The parasites were further cultured with different doses of corticosterone, dexamethasone or the vehicle for 5 days. [3H]Dehydroepiandrosterone (3H-DHEA) was added to the media and the cysticerci were further incubated for 6 or 24 h. Media were then removed and the steroids ether-extracted. Aliquots of the media were seeded on silica gel plates and developed in solvent systems. Parasites incubated in the presence of 3H-DHEA synthesized [3H]androstenediol, [3H]testosterone and [3H]17ß-oestradiol ([3H]17ß-E2). The addition of 100 nm or higher corticosterone doses to the media increased [3H]17ß-E2 synthesis fourfold after 24 h. Dexamethasone also increased [3H]17ß-E2 synthesis. The experiments presented here show for the first time that corticosterone and the synthetic GC dexamethasone modulate the synthesis of oestrogens by cysticerci.

Taenia crassiceps: chloride currents expressed in Xenopus oocytes upon injection of mRNA of cysticerci (WFU strain) isolated from mice.

To study the properties of ion channels of the tapeworm Taenia crassiceps, mRNA was isolated from cysticerci and injected into mature oocytes of the frog Xenopus laevis and ion currents were recorded four days after injection with the two-electrode voltage clamp technique. Oocytes injected with mRNA of T. crassiceps expressed outward currents (I(TC)) that activated instantly after onset of the test pulse, followed by a slow inactivation at potentials over +40 mV, with a reversal potential of -23.2+/-5 mV. They were not affected by changes on monovalent cationic composition of external media, but replacement of external chloride by gluconate shifted significantly the reversal potential, suggesting that I(TC) are anion currents, with a permeability sequence of NO3->Cl(-)>I(-)>>Gluconate. These currents were sensitive to changes of external pH but not to hypotonic challenges. They were significantly inhibited by DIDS, NPPB and Niflumic acid, but not by 9-anthracene. These results suggest that I(TC) are the result of expression of anion channels from the tapeworm T. crassiceps.

Immunolocalization of Taenia solium gap junction innexins.

In previous studies, ultrastructural observations revealed a large number of gap junctions (GJs) in the neck and immature proglottid tissues of Taenia solium tapeworms. In these helminths, cytoplasmic glycogen sacs are connected by numerous discrete GJs to other cells throughout the maturing strobilar tissue. Discontinuous sucrose gradients were used to purify membrane fractions containing GJs, which were identified by ultrastructural analysis. A trans-membrane peptide sequence from a highly conserved innexin region was used to construct a 20-amino acid synthetic peptide and used to raise polyclonal antibodies in rabbits that recognized both a 55 and a 67 kDa protein in a Western blot of the GJ-enriched pellet. Immunohistochemistry of larval and adult worm sections incubated with antiserum to the synthetic peptide and a secondary anti-rabbit IgG bound to fluorescein, revealed strong binding to the tegumentary surface of the worm, as well as patchy fluorescent areas in the parenchyma. The results indicate that both the tegument of cysticerci and adult T. solium contain innexin-rich membranes, which may function as a tegumentary transport system for small molecules.

Different effects of chorionic gonadotropin on Taenia crassiceps and Taenia solium cysticerci cultured in vitro.

Hormones play a significant role in murine Taenia crassiceps cysticercosis, and they may also participate in the susceptibility to Taenia solium cysticercosis. In the present study, in vitro effects are reported for human chorionic gonadotropin (hCG) on the larval stages of T. crassiceps (WFU strain) and T. solium. hCG effectively promotes parasite reproduction, i.e., it increases the number of buds on T. crassiceps cysticerci and the percentage of evagination and parasite length in T. solium. This is the first report in which a direct effect of hCG is reported for a parasite. hCG or mouse luteinizing hormone could be recognized by the cysticerci as mitogenic factors and contribute to the female and pregnancy bias toward susceptibility to T. crassiceps and T. solium cysticercosis, respectively.

Excitability changes in human forearm corticospinal projections and spinal reflex pathways during rhythmic voluntary movement of the opposite limb.

Rhythmic movements brought about by the contraction of muscles on one side of the body give rise to phase-locked changes in the excitability of the homologous motor pathways of the opposite limb. Such crossed facilitation should favour patterns of bimanual coordination in which homologous muscles are engaged simultaneously, and disrupt those in which the muscles are activated in an alternating fashion. In order to examine these issues, we obtained responses to transcranial magnetic stimulation (TMS), to stimulation of the cervicomedullary junction (cervicomedullary-evoked potentials, CMEPs), to peripheral nerve stimulation (H-reflexes and f-waves), and elicited stretch reflexes in the relaxed right flexor carpi radialis (FCR) muscle during rhythmic (2 Hz) flexion and extension movements of the opposite (left) wrist. The potentials evoked by TMS in right FCR were potentiated during the phases of movement in which the left FCR was most strongly engaged. In contrast, CMEPs were unaffected by the movements of the opposite limb. These results suggest that there was systematic variation of the excitability of the motor cortex ipsilateral to the moving limb. H-reflexes and stretch reflexes recorded in right FCR were modulated in phase with the activation of left FCR. As the f-waves did not vary in corresponding fashion, it appears that the phasic modulation of the H-reflex was mediated by presynaptic inhibition of Ia afferents. The observation that both H-reflexes and f-waves were depressed markedly during movements of the opposite indicates that there may also have been postsynaptic inhibition or disfacilitation of the largest motor units. Our findings indicate that the patterned modulation of excitability in motor pathways that occurs during rhythmic movements of the opposite limb is mediated primarily by interhemispheric interactions between cortical motor areas.

Ultrastructural damage of Trypanosoma cruzi epimastigotes exposed to decomplemented immune sera.

The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported, but the effects induced directly by immune serum depleted of complement remain unstudied. The aim of this work was to study the ultrastructural alterations induced in T. cruzi epimastigotes by immune mouse or rabbit sera with or without complement. A local isolate of T. cruzi (Queretaro) was used in all experiments. Immune sera were raised in both mouse and rabbit by immunization with T. cruzi epimastigote antigens. Light microscopy showed intense agglutination of epimastigotes when incubated with decomplemented mouse or rabbit immune sera. A distinctive ultrastructural feature of this agglutination pattern was the fusion of plasma membranes and a pattern of intercrossing between subpellicular microtubules. Agglutination was associated with fragmentation of nuclear membranes and swelling of cytoplasm, Golgi cisternae, endoplasmic reticulum, mitochondria and kinetoplast membranes. Agglutinated parasites also incorporated trypan blue stain. Results of [3H]-thymidine incorporation confirmed that epimastigotes exposed to specific antibodies in the absence of complement were incapable of proliferating. Ultrastructural changes observed in epimastigote micrographs incubated with decomplemented immune mouse sera were statistically significant (P<0.001) when compared with results obtained from images after incubation with decomplemented normal mouse sera.

Taenia solium: germinal cell precursors in tapeworms grown in hamster intestine.

BACKGROUND: In a previous study, it was shown that growth of evaginated metacestodes occurs in the germinative tissue of the neck by duplication of somatic stem cells. In these specimens, it was not possible to find the mitotic figures required to demonstrate duplication of germ cell lines. METHODS: Taenia solium strobilae were collected from the intestinal lumen of outbred hamsters infected orally with 10 metacestodes dissected from naturally infected pigs. Animals were anesthetized 1-10 days postinfection, the small intestine excised, submerged in PBS, and cut open longitudinally. Live Taenias were incubated for 6-8 h in medium containing colchicine or 3H-thymidine, washed, and embedded for electron microscopy. For light microscopy and autoradiography, longitudinal sections were cut from whole blocks and mounted on glass slides. A population of large cells without nuclear membranes and containing discrete aggregates of chromatin were observed apposed to myofibrils in the germinative tissue of the neck. These cells were confirmed by electron microscopy as metaphase mitotic figures, with chromosomes attached to a microtubular spindle, embedded in cytoplasm, without a nuclear membrane, and with characteristic centrioles. RESULTS: Only tapeworms in which 3H-thymidine was injected directly into the worm tissue by microsyringe were positive by autoradiography, demonstrating that in contrast to evaginated metacestodes, intestinal worms do not transport thymidine across the tegument. CONCLUSIONS: The results show that differentiating T. solium worms have a subset of stem cells that require passage through a mammalian host to go into mitosis, and that tapeworms grown in an experimental animal do not take up 3H-thymidine in vitro.

Formation of calcareous corpuscles in the lumen of excretory canals of Taenia solium cysticerci.

Platyhelminths, like many other organisms, are capable of producing mineral concretions. In cestodes these are referred to as calcareous corpuscles. Studies on these concretions in different cestodes both in vivo and in vitro have resulted in a number of hypotheses on their origin, formation, and structure. Calcareous corpuscles are believed to be of cellular origin, although the kind of cell involved and the mechanisms of mineralization remain under discussion. In the present paper we show that formation of calcareous corpuscles in cysticerci of Taenia solium is not of intracellular origin, as described for other cestodes, but occurs extracellularly in the lumen of protonephridial ducts in a way similar to that proposed for trematodes. This finding enhances the function of the protonephridial ducts, at least in the larvae of T. solium, to the roles formerly ascribed to the calcareous corpuscles.

Taenia solium: description of the intestinal implantation sites in experimental hamster infections.

Experimental infections in golden hamsters with viable Taenia solium metacestodes were used to study by light and electron microscopy the implantation site of the adult tapeworm in the intestinal wall. Implantation sites from 3-, 4-, 10-, and 40-day infections were located in the upper third of the duodenum, excised and fixed in Zenker's or Karnovsky's solution, embedded in Polybed resin, and sectioned longitudinally to observe the position of the worm on the intestinal wall. The scolex of the tapeworm was situated between host villi, with the rostellum penetrating the intestinal wall and the suckers entrapping adjacent villi. Serial sections through several whole implantation sites revealed that the worm was anchored to the host by all 4 suckers simultaneously, each of which was located at a different level and had entrapped intestinal villi in its cavity. Host tissue within the suckers was damaged, exhibiting various degrees of cell lysis and necrosis of epithelial and submucosal cells. The tegumentary surface and microtriches of the scolex were well preserved, with occasional coalescence of tegumentary microvesicles in 10- and 40-day-old infections; microtriches were in direct contact with the damaged host tissue. This study is the first morphological and ultrastructural description of the attachment of T. solium to the intestinal wall employing an experimental model, the results of which may contribute to a better understanding of the biology of human tapeworm infections.

Host cells in the spiral canal of Taenia solium (Cestoda) cysticerci.

The presence of host inflammatory cells inside the spiral canal of all viable Taenia solium cysts obtained from naturally infected pigs is described. Cells can penetrate into the vicinity of suckers and rostellum, although most appear damaged, suggesting that conditions in the canal are deleterious for them. These observations extend the localization of host inflammatory infiltrate to this intricate microniche, which may offer new approaches for the treatment of cysticercosis, based on a scolex-targeted action. The presence of host cells in the canal of cysts also poses the problem of the resulting contamination with host materials in studies using cysts extracts. As an example, host DNA contamination is readily detectable in genomic DNA isolated from T. solium and Taenia taeniaeformis cysts, as demonstrated by polymerase chain reaction amplification and subsequent sequencing of a segment of the 18S ribosomal gene.

Clinical manifestations and immunodiagnosis of gnathostomiasis in Culiacan, Mexico.

Gnathostomiasis was first described in Mexico in 1970, and endemic areas have been spreading in six states of this country. In Culiacan, Sinaloa, 300 cases of cutaneous larva migrans were recorded between January 1992 and December 1995. In addition, a Gnathostoma larva was surgically removed from the eye of one patient. Cutaneous lesions were observed mainly on the face, neck, arms, and legs. About 70% of the patients showed eosinophilia. A skin biopsy was carried out on 35 patients and the parasite was identified in histopathologic sections of 12 of these patients. In four patients, the larva migrated out spontaneously from the skin. An enzyme-linked immunosorbent assay using a crude somatic extract of adult Gnathostoma doloresi worms showed that 93% of the patients were seropositive, confirming the reliability of clinical diagnosis. A total of 14 advanced third-stage Gnathostoma larvae were found in four species of ichthyophagous birds captured on dams and dikes near the city of Culiacan. Scanning electron micrographs of human and bird larvae showed that they were morphologically indistinguishable from G. spinigerm. We conclude that the life cycle of Gnathostoma has been established in Sinaloa, and has become a serious public health issue for residents.

Autoradiographic analysis of the germinative tissue in evaginated Taenia solium metacestodes.

Evaginated Taenia solium metacestodes dissected from infected pork meat were incubated in vitro in RPMI 1640 medium with tritiated thymidine, washed, and further incubated for various chase periods. Worms were fixed and embedded in Poly/Bed and sections were processed for autoradiography. Results showed that all longitudinal sections had a germinative region located 500-700 mm posterior to the apex of the scolex with tegumentary cytons arranged in staggered columns perpendicular to the tegument. After 6-hr pulse and 0-12-hr chase periods, a large number of labeled cells were found in the parenchyma and tegumentary wall, included were myocytons, calcareous corpuscle cells, flame cells, osmoregulatory channel cells, and, in the medullary parenchyma, labeled undifferentiated round cells with a large nucleus, prominent nucleolus, abundant ribosomes, and no cytoplasmic organelles. These undifferentiated cells were not labeled after 24-hr and 48-hr chase periods, an observation that strongly suggests these cells divide and migrate toward the tegument in a pattern similar to that described for other cestodes. The morphology and localization of these cells support the view that they are stem cells that give rise to the various cell types of the tegumentary wall. The results indicate that T. solium contains a germinative tissue similar to that described in other cestodes, in which stem cells proliferate continuously, differentiate, and migrate to the tegument, constituting the main process by which these worms develop from metacestode to the adult stage.

Immunosuppression and inhibition of inflammation in mice induced by a small Taenia solium RNA-peptide to implanted T. solium metacestodes.

Subcutaneous implantation of Taenia solium metacestodes in mice induces an inflammatory reaction made up mainly of neutrophils and eosinophils after 12 days. Administration of a small RNA-peptide (metacestode factor, MF) purified from T. solium metacestodes significantly reduces the inflammatory site in both size and composition, yielding a very low number of eosinophils. The metacestodes implanted in control mice were completely destroyed and their remnants were surrounded by an intense inflammation predominantly made up of neutrophils and eosinophils. In contrast, metacestodes implanted in mice treated with MF showed apparently intact suckers, rostellum, hooks, and tegument. Inhibition of inflammation around the parasites was also observed in mice immunized with T. solium metacestode antigens and inoculated simultaneously with MF. Mice immunized only with T. solium metacestode antigens produced a granulomatous process around metacestodes that destroyed most of the large metacestode structures: suckers, rostellum, hooks, and tegument-wall tissues. Furthermore, treatment of mice with MF or implanted metacestodes decreased the antibody (P < 0.05) and cellular responses (P < 0.05) to metacestode antigens. The antibody responses was even lower when both of these treatments were given simultaneously. These findings support the idea that MF plays a key role in the down-regulation of the host immune response, contributing to the parasite's survival.