Molecular mechanisms involved in the differential effects of sex steroids on the reproduction and infectivity of Taenia crassiceps

The in vitro exposure of Taenia crassiceps cysticerci to 17-beta estradiol (E2) and progesterone (P4) stimulated their reproduction and infectivity. Testosterone (T4) and dihydrotestosterone (DHT) inhibited their reproduction and reduced their motility and infectivity. E2 and P4 increased, whereas T4 and DHT reduced, the expression of parasite c-fos and c-jun and DNA synthesis. In vitro exposure of cysticerci to sex steroids before their inoculation into recipient noninfected mice resulted in large parasite loads when pretreated with E2 and P4 and in smaller loads when pretreated with T4 and DHT To determine the possible molecular mechanisms by which sex steroids affect T. crassiceps, sex steroid receptors were amplified. Taenia crassiceps expressed estrogen receptors (both alpha and beta isoforms) and androgen receptors but no P4 receptors. These results demonstrate that sex steroids act directly on parasite reproduction by binding to a classic and specific sex steroid receptor on the parasite. The differential response of cysticerci to sex steroids may also be involved in their ability to grow faster in the murine female or feminized male host. This is the first report of direct sex steroid effects on the parasite possibly through sex steroid receptors in the cysticerci.     

Preferential growth of Taenia crassiceps cysticerci in female mice holds across several laboratory mice strains and parasite lines

A retrospective study of our 14-yr records on experimental Taenia crassiceps (ORF(fast) line) cysticercosis (n = 1,198) shows that in 16 of 17 different mice strains, female mice are more frequently infected and carry larger individual parasite loads than males. However, sexual differences in parasite loads significantly varies between strains in relation to their different genetic backgrounds (BALB > C57Bl = OTHERS > C3H). The coefficient of variation in all female mice is significantly smaller than that of all males, an indication of males' more potent, but erratically effective, restraint of cysticercus growth. Similar positive growth bias for female mice is shown by other lines of cysticerci, i.e., HYG(slow) and WFU(slow). These results contravene the usual expectation of female hosts being more resistant than males to parasite infections, and they point to the multiple factors that combined determine sex related differences of mice to experimental cysticercosis infection.     

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.     

Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci

Many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods and mammals. Cysticercosis produced by Taenia solium metacestodes is a widely distributed parasite infection that affects the human and the pig. Taenia crassiceps experimental murine cysticercosis has been used to explore the role of biological factors involved in host–parasite interactions. We had shown that T. crassiceps cysticercosis affects the serum concentration of steroid hormones and the reproduction behavior of the male mice host. In an effort to understand the biology of the parasite, we had investigated the parasite capacity to produce sex steroids. For this purpose, T. crassiceps cysticerci were incubated in the presence of different steroid precursors. TLC and recrystallization procedures showed that testosterone is produced from ³H-androstenedione in cysticerci. The conversion of ³H-testosterone to androstenedione, although present is much less significant. In addition, we had studied the production of testosterone by T. solium cysticerci. For this purpose, cysticerci were dissected from pork meat and incubated as above described. The results showed that T. solium cysticerci also produce testosterone. We have speculated about the importance of androgens in the growth of T. crassiceps cysticerci and found that the addition of the antiandrogen flutamide to the culture media of the parasites significantly decreased ³H-thymidine incorporation. We therefore hypothesized, that the ability of cysticerci to produce testosterone from steroid precursors might be important for the parasite growth and development.     

Infrapopulation dynamics of a wild strain of Taenia crassiceps (WFU) (Cestoda: Taeniidae) in BALB/cJ mice

Taenia crassiceps cysticerci form large infrapopulations that persist in the tissues of their rodent hosts. Early infrapopulation growth appears inhibited and is followed by rapid increases that appear not to be controlled by the host immune response. This investigation was undertaken to examine the infrapopulation growth dynamics of a normally developing strain (WFU) of T. crassiceps during a 60-day primary intraperitoneal (i.p.) infection. Three, 6, 9, 14, 28, and 60 days after i.p. inoculation of 5 cysticerci, mice were killed, and the numbers of larvae, developmental stage, and buds per larva were recorded. Larval infrapopulation abundance increased exponentially beginning on day 6 postinoculation (PI), indicating an initial lag in reproduction. A stage-structured exponential growth model, assuming no mortality, fits the larval infrapopulation dynamics in terms of the numbers of larvae in reproductive and nonreproductive stages, indicating that cysticerci evade or suppress (or both) host immune mechanisms that are parasite restrictive after the first week of infection.     

Kinetics and characterization of cellular responses in the peritoneal cavity of mice infected with Taenia crassiceps.

Changes in the leukocyte population of the peritoneal cavity ensue immediately after infection with Taenia crassiceps metacestodes. Basophils and neutrophils decrease, whereas macrophages, monocytes, and lymphocytes increase to reach only modest levels by 6 wk and then diminish to nearly disappear by 15 wk when the parasite begins rapid reproduction. Eosinophils also appear early in infection, but then abate to lower levels that persist. In late infections, when the mass of cysticerci equals that of the mouse, the cysticerci grow among surprisingly few inflammatory cells. Mingling with the peritoneal inflammatory cells is a number of odd-looking cells that could correspond to the metaplasic mesothelial cells of the host or be of parasite origin. These cells are multinucleated, they aggregate in varigerated clusters, and form cystic structures in vitro; they also bind specific anti-T. crassiceps antibodies and specific T. crassiceps DNA probes in their nuclei. When the peritoneal cell exudate is reinjected intraperitoneally into naive mice, the odd-looking cells subsist for months, inducing in the host the synthesis of specific anti-T. crassiceps antibodies and immune resistance to challenge but do not reassemble into cysticerci even after 6 mo of inoculation. The early appearance and the immunogenic and antigenic properties of these odd-looking cells suggest they are important protagonists in the early host-parasite confrontation when the outcome of infection is set.     

Immunological mediation of gonadal effects on experimental murine cysticercosis caused by Taenia crassiceps metacestodes.

Female BALB/c mice are naturally more susceptible than males to intraperitoneal experimental infection with Taenia crassiceps metacestodes. Gonadectomy tends to equalize susceptibility between sexes by reducing in half the mean individual intensity of females and by tripling that of males. The effect of gonadectomy is seen only in mice with intact immune systems but not in irradiated mice. Purified sex hormones (17-beta estradiol, testosterone, and progesterone) do not affect cysticercus reproduction or growth in vitro. Thus, gonadal effect on mouse susceptibility to cysticercosis appears to be mediated via the immune system, and it is probably not the consequence of the major sex steroids acting directly upon the parasites. Because sublethal irradiation increases the intensity in gonadectomized females and intact males, whereas that of gonadectomized males and intact females remains unchanged, irradiation results are consistent with the hypothesis that immunological events that participate in controlling the growth of cysticerci are inhibited by ovaries and stimulated by testes.     

Fatal cysticercosis by Taenia crassiceps (Cyclophyllidea: Taeniidae) in a presumed immunocompromised canine host

Cysticercosis in a canine host (Canis familiaris) attributable to the taeniid cestode Taenia crassiceps is reported for the first time in North America. Numerous parent and daughter cysticerci occurred in a massive intrapleural and intraperitoneal infection in an apparently immunocompromised host. The largest cysticerci were ovoid to elongate, 5-9 mm in maximum length, and armed with 32-34 rostellar hooks in 2 rows; small hooks measured 114-143 microm long (x = 124+/-8.2 microm), and large hooks were 156-180 microm (x = 163+/-7.4 microm). Taenia crassiceps is widespread in boreal North America and, like a number of other taeniids, constitutes a potential risk as a zoonotic parasite. The immunological status of the host may be important in determining the outcome of infections for this and other taeniids in atypical hosts.     

Histomorphological study of the preputial and clitoral glands in BALB/c mice with experimental Taenia crassiceps infections

Male preputial and female clitoral glands of mice undergo development that depends on the level of hormones in the animal. Experimental infection with Taenia crassiceps cysticerci results in significant physiological modifications in the host. Here, we investigated the histomorphological alterations induced by the parasite in these pheromonal glands. Preputial and clitoral glands were recovered from mice at 15, 35, 50, and 70 days postinfection (DPI). The glands were examined macroscopically and microscopically after histological preparation. Male preputial glands show a marked atrophy 35 days after infection. This atrophy is the result of a disorganization of the acinus tissue structure. During the course of infection, the basal, intermediate, and mature acinar cell layers are reduced, and finally, at 70 DPI, the gland includes only the duct system and fibrotic structures. In contrast, females are not affected by the infection because no modifications were observed in the morphology or histology of the clitoral glands. A probable cause for such a divergence between infected male and female mice might be related to a sex steroid imbalance as described during T. crassiceps infection.     

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.     

Identification of loci controlling restriction of parasite growth in experimental Taenia crassiceps cysticercosis

Human neurocysticercosis (NC) caused by Taenia solium is a parasitic disease of the central nervous system that is endemic in many developing countries. In this study, a genetic approach using the murine intraperitoneal cysticercosis caused by the related cestode Taenia crassiceps was employed to identify host factors that regulate the establishment and proliferation of the parasite. A/J mice are permissive to T. crassiceps infection while C57BL/6J mice (B6) are comparatively restrictive, with a 10-fold difference in numbers of peritoneal cysticerci recovered 30 days after infection. The genetic basis of this inter-strain difference was explored using 34 AcB/BcA recombinant congenic strains derived from A/J and B6 progenitors, that were phenotyped for T. crassiceps replication. In agreement with their genetic background, most AcB strains (A/J-derived) were found to be permissive to infection while most BcA strains (B6-derived) were restrictive with the exception of a few discordant strains, together suggesting a possible simple genetic control. Initial haplotype association mapping using >1200 informative SNPs pointed to linkages on chromosomes 2 (proximal) and 6 as controlling parasite replication in the AcB/BcA panel. Additional linkage analysis by genome scan in informative [AcB55xDBA/2]F1 and F2 mice (derived from the discordant AcB55 strain), confirmed the effect of chromosome 2 on parasite replication, and further delineated a major locus (LOD = 4.76, p<0.01; peak marker D2Mit295, 29.7 Mb) that we designate Tccr1 (T. crassiceps cysticercosis restrictive locus 1). Resistance alleles at Tccr1 are derived from AcB55 and are inherited in a dominant fashion. Scrutiny of the minimal genetic interval reveals overlap of Tccr1 with other host resistance loci mapped to this region, most notably the defective Hc/C5 allele which segregates both in the AcB/BcA set and in the AcB55xDBA/2 cross. These results strongly suggest that the complement component 5 (C5) plays a critical role in early protective inflammatory response to infection with T. crassiceps.     

Inhibitory role of antibodies in the development of Taenia solium and Taenia crassiceps toward reproductive and pathogenic stages.

Untreated Taenia solium cysticerci obtained from different naturally infected pigs vary notably in their capacity to develop into intestinal tapeworms in prednisolone-treated hamsters, whereas cells derived from Taenia crassiceps cysticerci after 2 mo of infection almost always develop to cysticerci in the peritoneal cavity of susceptible BALB/cAnN mice. Preincubation of whole cysticerci or parasite cells with mice immunoglobulins raised against an 18-mer peptide epitope (GK-1) common to both parasites significantly interferes with both transformations. These crippling effects of antiparasite antibodies suggest new forms of immunological interference with parasite biology other than simple killing. Antibodies that cripple biological functions of the parasite, e.g., their development to reproductive or pathogenic stages, make them important protagonists in taeniasis/cysticercosis disease as classic parasitocidal antibodies. Different serum levels of crippling antibodies in the infected pigs could be responsible for the varied ability of cysticerci to convert to tapeworms. Antigens capable of inducing crippling antibodies, e.g., GK-1, could be useful as a therapeutic vaccine for pigs in order to reduce parasite transmission.     

Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci

Many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods and mammals. Cysticercosis produced by Taenia solium metacestodes is a widely distributed parasite infection that affects the human and the pig. Taenia crassiceps experimental murine cysticercosis has been used to explore the role of biological factors involved in host–parasite interactions. We had shown that T. crassiceps cysticercosis affects the serum concentration of steroid hormones and the reproduction behavior of the male mice host. In an effort to understand the biology of the parasite, we had investigated the parasite capacity to produce sex steroids. For this purpose, T. crassiceps cysticerci were incubated in the presence of different steroid precursors. TLC and recrystallization procedures showed that testosterone is produced from ³H-androstenedione in cysticerci. The conversion of ³H-testosterone to androstenedione, although present is much less significant. In addition, we had studied the production of testosterone by T. solium cysticerci. For this purpose, cysticerci were dissected from pork meat and incubated as above described. The results showed that T. solium cysticerci also produce testosterone. We have speculated about the importance of androgens in the growth of T. crassiceps cysticerci and found that the addition of the antiandrogen flutamide to the culture media of the parasites significantly decreased ³H-thymidine incorporation. We therefore hypothesized, that the ability of cysticerci to produce testosterone from steroid precursors might be important for the parasite growth and development.     

Taenia crassiceps: host treatment alters glycolisis and tricarboxilic acid cycle in cysticerci

Human cysticercosis by Taenia crassiceps is rare although it is considered of zoonotic risk, especially to immunocompromised individuals. Albendazole and praziquantel are widely used and effective in its treatment. Their active forms inhibit the glucose uptake by the parasite and induce muscle contractions that alter its glycogen levels interfering in the energetic metabolism of the parasite and leading to its death. The aim of this study was to evaluate alterations in glycolysis, the tricarboxylic acid cycle and glucose concentrations caused by low dosage treatments of the hosts with albendazole and praziquantel. Therefore, T. crassiceps intraperitoneally infected mice were treated by gavage feeding with 5.75 or 11.5 mg/kg of albendazole and 3.83 or 7.67 mg/kg of praziquantel. The treated mice were euthanized after 24 h and the cysticerci collected were morphologically classified into initial, larval or final phases. Concentrations of the organic acid produced and glucose were evaluated to detect alterations into the glycolysis and the tricarboxylic acid cycle pathways through chromatography and spectrophotometry. The low dosage treatment caused a partial blockage of the glucose uptake by the cysticerci in spite of the non significant difference between its concentrations. An activation of the tricarboxylic acid cycle was noted in the cysticerci that received the treatment due to an increase in the production of citrate, malate and α-ketoglutarate and the consumption of oxaloacetate, succinate and fumarate. The detection of α-ketoglutarate indicates that the cysticerci which were exposed to the drugs after host treatment present different metabolic pathways than the ones previously described after in vitro treatment.     

The growth of the metacestodes of Taenia crassiceps in white mice.

The effect of the sex of the intermediate host on the growth and development of the metacestodes of Taenia crassiceps has been investigated. Three different strains of mice were used, the C R S, L A C A and C F L P strains and two strains of metacestode, the Toi and the E R S strains. The results show that matacestodes of T. crassiceps grow and multiply more rapidly in female than in male mice, although both sexes are equally susceptible to the infection. The origin of the parasite, i.e., from a rat or mouse host, affects the parasite growth.     

Parasite regulation by host hormones: an old mechanism of host exploitation?

Recent experimental evidence suggests that parasites can not only evade immune responses actively but also exploit the hormonal microenvironment within the host to favor their establishment, growth and reproduction. The benefit for parasites of hormonal exploitation is so great that they have evolved structures similar to the steroid and protein hormone receptors expressed in upper vertebrates that can bind to the hormonal metabolites synthesized by the host. This strategy is exemplified by two parasites that respond to adrenal steroids and sexual steroids, respectively: Schistosoma mansoni and Taenia crassiceps. Understanding how the host endocrine system can, under certain circumstances, favor the establishment of a parasite, and characterizing the parasite hormone receptors that are involved might aid the design of hormonal analogs and drugs that affect the parasite exclusively.     

Taenia crassiceps cysticercosis: protective effect and immune response elicited by DNA immunization

The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci present in all stages of Taenia solium (KETc7), cloned into pcDNA3 plasmid with the signal peptide sequence of the beta-glycan receptor (pTc-sp7), has been shown to be effective in protecting mice against experimental infection of T. crassiceps. To explore further the possibilities of this form of immunization and the immune response induced, mice were injected intramuscularly (i.m.) or intradermally (i.d.) with 3 doses of pTc-sp7. Similar levels of resistance were found using either i.m. or i.d. immunization. Spleen cells from i.d. and i.m. DNA immunized mice induced a specific T-cell response to T. crassiceps antigens and to a synthetic peptide from the immunogen itself (GK-1). Proliferated cells were especially enriched in CD8+ CD4- T-lymphocytes. A clear increase in the percentage of CD3+ cells that produce gamma-interferon and interleukin-2 was detected when measuring the intracellular cytokine production, an indication of the pTc-sp7 capacity to induce an effective cellular response. These results provide encouraging information on the use of KETc7 in the prevention of cysticercosis as well as a first insight into the characterization of the immune response induced by pTc-sp7 that hints to the relevance of cellular immunity in protection.     

Regulation of the immune response to cestode infection by progesterone is due to its metabolism to estradiol

The aim of this work was to investigate the role of progesterone during Taenia crassiceps cysticercosis, and the immunological mechanisms involved in its effects, by relating progesterone treatment to whole parasite counts, to host humoral and cellular immune response, to the presence or absence of nuclear receptors to sex steroids in splenocytes, and to serum sex steroid levels in infected mice of both genders. Progesterone treatment increased parasite loads two-fold in females and three-fold in males compared with control mice. The expression of the Th2 cytokine profile (IL-4, IL-6 and IL-10) was markedly increased in infected mice of both genders, while progesterone treatment returned this expression to basal levels. However, the Th1 cytokine profile (IFN-gamma and TNF-alpha) was not affected by infection, whilst progesterone treatment increased the expression of both cytokines two-fold compared to uninfected, infected and placebo-treated mice. Testosterone serum levels decreased in infected male mice by 95%, and treatment with progesterone did not affect them. In females, no change in testosterone levels was observed. Progesterone levels increased three-fold only in progesterone-treated infected mice of both sexes, while estradiol levels in female and male progesterone-treated infected mice increased two-fold compared to infected control mice. The infection markedly induced the expression of progesterone receptor (PR) isoforms A and B in splenocytes of infected mice of both genders (five-fold). Metabolism of progesterone to estradiol was demonstrated by the use of the anti-estrogen tamoxifen, which reduced parasite loads 100% in infected mice of both sexes treated with progesterone. These results suggest that progesterone, possibly through its metabolism to estradiol, affects establishment, growth and reproduction of the helminth parasite T. crassiceps.     

Tamoxifen treatment induces protection in murine cysticercosis

Administration of tamoxifen (an antiestrogen) produced an 80% parasite load reduction in female mice, and a weaker effect of 50% in male mice. This protective effect was associated in both sexes, with an increase in the mRNA levels of interleukin (IL)-2 (a cytokine associated with protection against cysticerci) and IL-4 (no effect on infection). tamoxifen treatment modified 17-beta estradiol production in females, whereas serum testosterone was not affected. However, the expression of the 2 types of estrogen receptor (ER), i.e., ER-alpha and ER-beta, in the spleen of infected mice of both sexes, was decreased by tamoxifen treatment. In vitro, treatment of Taenia crassiceps with tamoxifen reduced reproduction and loss of motility. These results indicate that tamoxifen treatment is a new therapeutic possibility to treat cysticercosis, because it can act at both ends of the host-parasite relationship, i.e., by increasing the cellular immune response protective against the parasite and by directly affecting the parasite's reproduction and survival.