**4. The case of Cestodes**

276 Sex Steroids

by which sex steroids may exert their biological effects involves interactions with either cytoplasmic or nuclear receptors. For these hormones to have an effect on immune system cells, the presence of hormone receptors in these cells is necessary. Although steroid hormones also exert effects by non-genomic mechanisms by acting on cell surface receptors and triggering signaling cascades, it is currently accepted that the main route of biological activity occurs by means of specific nuclear receptors (NR) that function as transcription factors, and coordinate, after binding to their ligand, the expression of target genes. The following NR are mediators of these effects: estrogen receptors (ER) and androgen receptor (AR); the estrogen receptors ER- and ER-, each coded for by an individual gene, whose predominating ligand is 17β-estradiol; progesterone receptor (PR), which has variants A and B generated from the same gene by alternative splicing, and whose main ligand is progesterone. Androgen receptors (AR), coded for by a single gene and its ligands being testosterone and dehydrotestosterone (DHT). In fact, the binding between estradiol (E2) and its membrane ER activates group I and II of the metabotropic glutamate receptor (Boulware et al., 2005). It should here be mentioned that ER is able to bind to Src kinases through their highly conserved SH2 domains, which could considerably modify the effect of ERK 1/2 on the phosphorylation pattern of this transcription factor (Auricchio et al., 2008). Recently, three new putative membrane progesterone receptors (mPRs), mPRα, mPRβ, and mPRγ have been described and detected also on T lymphocytes (Dosiou et al., 2008). The mechanism of action of these membrane progesterone receptors is suggested to be through Gi-protein activation. Previous findings have also revealed unconventional non-genomic surface receptors for testosterone in rat T cells. These belong to the class of membrane receptors coupled to phospholipase C (PLC) via a pertussis toxin-sensitive G-protein. Binding of testosterone to these cell surface receptors causes a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) and an increased formation of inositol 1,4,5-triphosphate and diacylglycerol (Benten et al., 1999). Preliminary evidence indicates that in murine T

, presumably due to Ca2+ influx

cells, testosterone also induces a rapid rise in [Ca2+]i

immunoregulators (Verthelyi, 2001).

triggered by binding of testosterone to receptors on the outer surface of T cells.

Sexual dimorphism (SD) in parasitic infections it is a scarcely studied biological phenomenon of considerable significance for individual health as well as for the evolution of species. Most of the poor knowledge about this topic is related to the wrong concept of the female supremacy in infectious diseases. However, there are many notable exceptions to this rule of a favorable female bias in susceptibility to infection. Particularly in the host's sexual differences to cystercosis infection, females are more likely to become infected, to carry larger parasite loads, to be more severely affected and more reticent to develop protective immunity to variable degrees that associate with their genetic backgrounds and times of infection. The importance of the interaction between the immune and endocrine systems becomes evident in particular circumstances of the lifespan of an organism, such as pregnancy, autoimmune diseases, and some time, it is also affected by infectious diseases. In all cases, the available evidence underscores the importance of sex steroids as

The hormonal microenvironment and in particular the balance of male and female hormones may favor survival of certain parasites under certain circumstances. Predominance of a sex-distinctive steroid may directly induce reproduction, growth or

**3. Sex steroids and immune response to helminthes parasites** 

Cestoda is the class of parasitic flatworms, commonly called tapeworms that live in the digestive tract of vertebrates as adults and often in the bodies of various animals as juveniles. There are two subclasses in class Cestoda, the Cestodaria and the Eucestoda. By far the most common and widespread are the Eucestoda, with only a few species of unusual worms in subclass Cestodaria. The cyclophyllideans are the most important to humans because they infect people and livestock. Two important tapeworms are the pork tapeworm, *Taenia solium*, and the beef tapeworms, *T. saginata* (Morales-Montor et al., 2004). Taennids, particularly *Taenia solium* (causal agent of porcine cysticercosis and human neurocysticercosis) and *Taenia crassiceps* (causal agent of murine cysticercosis) are highly evolved parasites that have developed diverse mechanisms of survival that facilitate their establishment in the hosts. Taennids can also exploit the hormonal microenvironment within the host in their favor (Escobedo et al., 2005; Locksley, 1997). Taennids have evolved structures similar to the steroid and protein hormone receptors expressed in upper vertebrates, with binding properties and terminal effects similar to the hormonal metabolites synthesized by the host (Damian, 1989; Salzet et al., 2000). In the next paragraphs, we summarize the findings on the role of sex steroids in two cestodes: *Taenia crassiceps* and *Taenia solium*.

#### **4.1 Experimental** *T. solium* **Taeniosis/cysticercosis**

*T. solium* is an ancient parasite that still threatens public health and porcine husbandry in Latin America, Africa and Asia, and is re-emerging in developed countries on account of the massive human migrations of modern times (Hoberg, 2002; DeGiorgio et al., 2005; Sorvillo et al., 2011). Cysticercosis results from the ingestion of the *T. solium* eggs by intermediate hosts (humans and pigs, principally), to then hatch in the intestines liberating motile oncospheres that penetrate the circulation and distribute in the organism. Oncospheres may establish in muscles, subcutaneous connective tissue, central nervous system, liver, and other organs of the host, where they develop into cysticerci, the larvae of *T. solium* contained within a vesicular translucent structure of about one cm in size (Sciutto et al., 2000). Once developed, many cysticerci die leaving scar tissue or nodular calcifications, while others live-on causing chronic and severe organic malfunction because of space-occupation and/or local inflammation, especially when located in the brain of the human intermediate host. Humans are also the only definitive hosts of the intestinal adult tapeworm, the stage in which the parasite is capable of sexual reproduction and of massive egg production. Pigs are nowadays the preferred intermediary host for the *T. solium*'s larval stage (i.e., cysticercus), a necessary

The Role of Sex Steroids in the Host-Parasite Interaction 279

to both vehicle-treated and non-manipulated infected animals. In contrast to control and vehicle groups, progesterone diminished tapeworm length by 75% and increased proliferation rate of leukocytes from spleen and mesenteric lymph nodes of infected hamsters by 5-fold. IL-4, IL-6 and TNF- expression at the duodenal mucosa, promoted

The issue of sexual dimorphism in naturally acquired porcine cysticercosis is a bit stronger than it is in human cysticercosis. Male rural pigs castrated 4 months before sacrifice show a cysticercosis prevalence double that of non-castrated male pigs, and so do pregnant sows (Morales et al., 2002; 2006). There is, however, an exposure catch obscuring the reason of such associations: castrated pigs flock around a dominant female that effectively search for foodstus in the open fields whilst sexually active boars are almost wild creatures, roaming around marginally to any flock and are thus presumed to be less well nurtured and less exposed to consuming human feces and becoming infected. Anyways, research is in progress to ascertain if any endocrinological changes in the sex steroids of rural pigs associate with cysticercosis. Moreover, frequency of *T. solium* pig cysticercosis is increased during pregnancy, when there is a significant increase in progesterone levels (Morales et al., 2002; Peña et al., 2007). It has also been demonstrated that castration in naturally infected male boars, induces an increase in the prevalence of cysticercosis, which highlights the possible role of host androgens to restrict parasite establishment and estrogens to facilitate it

The effects of progesterone and its antagonist RU486, on scolex evagination, which is the initial step in the development of the adult worm, have been demonstrated (Escobedo, et al, 2010). Interestingly, progesterone increased *T. solium* scolex evagination and worm growth, in a concentration-independent pattern. Progesterone effects could be mediated by a novel *T. solium* progesterone receptor (TsPR), since RU486 inhibits both scolex evagination and worm development induced by progesterone. By using RT-PCR and western blot, sequences related to progesterone receptor were detected in the parasite. A phylogenetic analysis reveals that TsPR is highly related to fish and amphibian progesterone receptors, whereas it has a distant relation with birds and mammals. Conclusively, progesterone directly acts upon *T. solium* cysticerci, possibly through its binding to a progesterone receptor

Due to the intrinsic difficulties in working with the natural hosts (pigs and humans) of *T. solium* or because of the high costs of sufficient pigs plus the slowness in data retrieval, we have used an experimental cysticercosis approach to gain knowledge of the complex host (H) parasite (P) relationship in cysticercosis. Murine intraperitoneal cysticercosis is caused by the taenid *Taenia crassiceps* and it has been useful to explore the physiological host factors associated with porcine cysticercosis, and to some degree, with human neurocysticercosis (Sciutto et al., 2011). Intraperitoneal *T. crassiceps* cysticercosis of mice lends itself well to controlled and reproducible experimentation, generating numerical data of parasite loads in individual mice in a matter of weeks after infection. Its general representation of other forms of cysticercosis has later been strengthened by similar results in other mouse and parasite strains, by the parasite's extensive sharing of antigens with other taennids and cestodes and

local exacerbation of inflammatory infiltrate.

**4.2 Sexual steroids directly act upon** *Taenia solium*

synthesized by the parasite (Escobedo et al, 2010).

**4.3 Experimental** *Taenia crassiceps* **murine cysticercosis** 

(Morales et al., 2002).

stage in the parasite´s life cycle before its eventual transformation into an adult tapeworm upon their ingestion by humans in infected and uncooked pork meat.

Host's sexual dimorphism in *Taenia solium* infections is much less obvious than that of experimental *Taenia crassiceps* cysticercosis in laboratory mice. Nonetheless, there are various hints pointing in that direction and several speculations as to the reasons behind such low profiles of sexual differences in cysticercotic pigs and humans. Human's immunological contact with any of *T. solium*'s developmental forms is likely to induce an immune response. The high mean prevalence of seropositive individuals in the open population of Mexico (~1%) (Larralde et al., 1992), when compared with the presumed prevalence of neurocysticercosis (~0.1 to 10%) (Fleury et al., 2003; 2006), is taken to indicate that very few of the contacts result in the establishment of the cysticercus in the tissues of humans and less so in their brains. In view of the many weaknesses of such numbers and assumptions lacking the objective finding of the parasite in the suspected human cases it's somewhat adventurous to interpret them as signs of sexual dimophism in human disease, even if significant differences in serological prevalence favor women over men in Mexico (Larralde et al., 1992). The more so if no credible prevalence differences of neurocysticercosis were supported by necropsy studies (Rabiela et al., 1982). Nonetheless, there are other findings indicating to sexual dimorphism among human neurocyticercotic patients. Women develop more frequently generalized encephalitis than men and, when bearing subarchnoidal and ventricular vesicular parasites, women show higher inflammatory profiles in their cerebral spinal fluid than men (Del Brutto et al., 1988; Fleury et al., 2004). However, in those same patients there were no other immunological signs of sexual dimorphism (Chavarria et al., 2005). Also, in Peru, it was reported that subjects included a newly devised anti-helminth treatment protocol were mostly women highly suspected of carrying an adult *T. solium* in their intestines. Likewise, women were recently reported to harbor more single cysticercotic calcified lesions in their right cerebral hemisphere than in the left. Presumably, lateralization of calcified cysticerci reflects the differential immunological abilities between the cerebral hemispheres (Meador et al., 2004). Although no sexual differences in these cerebral abilities have been notified, cysticercus lateralization is not found in male neurocysticercotic patients. Gender discriminatory practices in health services also throw shadows upon morbidity and mortality differences between women and men in Mexico: men request more frequently medical attention at hospitals than women do, yet they have similar mortality rates in the many diseases not in close association with genital organs, including infections. It would then follow that Mexican women more frequently resort to informal consult and therapy of their ailments than men do, thus defaulting from morbidity data and appearing for consult in the final stages of disease. Notwithstanding the many sex and gender associated biological and social biases involved, the findings of sexual dimorphism in cysticercotic humans should not be confidently dismissed.

Sex steroids play an important role during *T. solium* infection, particularly progesterone has been proposed as a key immunomodulatory hormone involved in susceptibility to human taeniosis in woman and cysticercosis in pregnant pigs. Then, we evaluated the effect of progesterone administration upon experimental taeniosis in hamsters (*Mesocricetus auratus*). Intact female adult hamsters were randomly divided into 3 groups: progesteronesubcutaneously treated; olive oil-treated, as the vehicle group; and untreated controls. Animals were treated every other day during 4 weeks. After 2 weeks of treatment, all hamsters were orally infected with 4 viable *T. solium* cysticerci. After 2 weeks post infection, progesterone-treated hamsters showed reduction in adult worm recovery by 80%, compared

stage in the parasite´s life cycle before its eventual transformation into an adult tapeworm

Host's sexual dimorphism in *Taenia solium* infections is much less obvious than that of experimental *Taenia crassiceps* cysticercosis in laboratory mice. Nonetheless, there are various hints pointing in that direction and several speculations as to the reasons behind such low profiles of sexual differences in cysticercotic pigs and humans. Human's immunological contact with any of *T. solium*'s developmental forms is likely to induce an immune response. The high mean prevalence of seropositive individuals in the open population of Mexico (~1%) (Larralde et al., 1992), when compared with the presumed prevalence of neurocysticercosis (~0.1 to 10%) (Fleury et al., 2003; 2006), is taken to indicate that very few of the contacts result in the establishment of the cysticercus in the tissues of humans and less so in their brains. In view of the many weaknesses of such numbers and assumptions lacking the objective finding of the parasite in the suspected human cases it's somewhat adventurous to interpret them as signs of sexual dimophism in human disease, even if significant differences in serological prevalence favor women over men in Mexico (Larralde et al., 1992). The more so if no credible prevalence differences of neurocysticercosis were supported by necropsy studies (Rabiela et al., 1982). Nonetheless, there are other findings indicating to sexual dimorphism among human neurocyticercotic patients. Women develop more frequently generalized encephalitis than men and, when bearing subarchnoidal and ventricular vesicular parasites, women show higher inflammatory profiles in their cerebral spinal fluid than men (Del Brutto et al., 1988; Fleury et al., 2004). However, in those same patients there were no other immunological signs of sexual dimorphism (Chavarria et al., 2005). Also, in Peru, it was reported that subjects included a newly devised anti-helminth treatment protocol were mostly women highly suspected of carrying an adult *T. solium* in their intestines. Likewise, women were recently reported to harbor more single cysticercotic calcified lesions in their right cerebral hemisphere than in the left. Presumably, lateralization of calcified cysticerci reflects the differential immunological abilities between the cerebral hemispheres (Meador et al., 2004). Although no sexual differences in these cerebral abilities have been notified, cysticercus lateralization is not found in male neurocysticercotic patients. Gender discriminatory practices in health services also throw shadows upon morbidity and mortality differences between women and men in Mexico: men request more frequently medical attention at hospitals than women do, yet they have similar mortality rates in the many diseases not in close association with genital organs, including infections. It would then follow that Mexican women more frequently resort to informal consult and therapy of their ailments than men do, thus defaulting from morbidity data and appearing for consult in the final stages of disease. Notwithstanding the many sex and gender associated biological and social biases involved, the findings of sexual dimorphism

upon their ingestion by humans in infected and uncooked pork meat.

in cysticercotic humans should not be confidently dismissed.

Sex steroids play an important role during *T. solium* infection, particularly progesterone has been proposed as a key immunomodulatory hormone involved in susceptibility to human taeniosis in woman and cysticercosis in pregnant pigs. Then, we evaluated the effect of progesterone administration upon experimental taeniosis in hamsters (*Mesocricetus auratus*). Intact female adult hamsters were randomly divided into 3 groups: progesteronesubcutaneously treated; olive oil-treated, as the vehicle group; and untreated controls. Animals were treated every other day during 4 weeks. After 2 weeks of treatment, all hamsters were orally infected with 4 viable *T. solium* cysticerci. After 2 weeks post infection, progesterone-treated hamsters showed reduction in adult worm recovery by 80%, compared to both vehicle-treated and non-manipulated infected animals. In contrast to control and vehicle groups, progesterone diminished tapeworm length by 75% and increased proliferation rate of leukocytes from spleen and mesenteric lymph nodes of infected hamsters by 5-fold. IL-4, IL-6 and TNF- expression at the duodenal mucosa, promoted local exacerbation of inflammatory infiltrate.

The issue of sexual dimorphism in naturally acquired porcine cysticercosis is a bit stronger than it is in human cysticercosis. Male rural pigs castrated 4 months before sacrifice show a cysticercosis prevalence double that of non-castrated male pigs, and so do pregnant sows (Morales et al., 2002; 2006). There is, however, an exposure catch obscuring the reason of such associations: castrated pigs flock around a dominant female that effectively search for foodstus in the open fields whilst sexually active boars are almost wild creatures, roaming around marginally to any flock and are thus presumed to be less well nurtured and less exposed to consuming human feces and becoming infected. Anyways, research is in progress to ascertain if any endocrinological changes in the sex steroids of rural pigs associate with cysticercosis. Moreover, frequency of *T. solium* pig cysticercosis is increased during pregnancy, when there is a significant increase in progesterone levels (Morales et al., 2002; Peña et al., 2007). It has also been demonstrated that castration in naturally infected male boars, induces an increase in the prevalence of cysticercosis, which highlights the possible role of host androgens to restrict parasite establishment and estrogens to facilitate it (Morales et al., 2002).

#### **4.2 Sexual steroids directly act upon** *Taenia solium*

The effects of progesterone and its antagonist RU486, on scolex evagination, which is the initial step in the development of the adult worm, have been demonstrated (Escobedo, et al, 2010). Interestingly, progesterone increased *T. solium* scolex evagination and worm growth, in a concentration-independent pattern. Progesterone effects could be mediated by a novel *T. solium* progesterone receptor (TsPR), since RU486 inhibits both scolex evagination and worm development induced by progesterone. By using RT-PCR and western blot, sequences related to progesterone receptor were detected in the parasite. A phylogenetic analysis reveals that TsPR is highly related to fish and amphibian progesterone receptors, whereas it has a distant relation with birds and mammals. Conclusively, progesterone directly acts upon *T. solium* cysticerci, possibly through its binding to a progesterone receptor synthesized by the parasite (Escobedo et al, 2010).

#### **4.3 Experimental** *Taenia crassiceps* **murine cysticercosis**

Due to the intrinsic difficulties in working with the natural hosts (pigs and humans) of *T. solium* or because of the high costs of sufficient pigs plus the slowness in data retrieval, we have used an experimental cysticercosis approach to gain knowledge of the complex host (H) parasite (P) relationship in cysticercosis. Murine intraperitoneal cysticercosis is caused by the taenid *Taenia crassiceps* and it has been useful to explore the physiological host factors associated with porcine cysticercosis, and to some degree, with human neurocysticercosis (Sciutto et al., 2011). Intraperitoneal *T. crassiceps* cysticercosis of mice lends itself well to controlled and reproducible experimentation, generating numerical data of parasite loads in individual mice in a matter of weeks after infection. Its general representation of other forms of cysticercosis has later been strengthened by similar results in other mouse and parasite strains, by the parasite's extensive sharing of antigens with other taennids and cestodes and

The Role of Sex Steroids in the Host-Parasite Interaction 281

IL-2 and IFN-γ in both sexes, while the secretion of cytokines involved in the specific humoral response (IL-6, IL-10 and IL-4) is enhanced (Terrazas et al., 1998, 1999). Thus, striking differences in susceptibility to cysticercosis between male and female mice may involve the joint action of the immune system and the gonads, both driven by a parasite that is able to change the parasite's restrictive male normal hormonal milieu during chronic

To strengthen the above notions and in an effort to identify the sex steroids involved we studied the effects of testosterone, dihydrotestosterone, and 17β-estradiol in castrated mice of both sexes infected with *Taenia crassiceps* cysticerci (Morales-Montor et al., 2002a). In this study, we found that castration and treatment with either testosterone or dihydrotestosterone before infection markedly decreased parasite loads in both gender mice, while the treatment with 17β-estradiol increased it in both genders (Morales-Montor et al., 2002a). The specific splenocyte cell proliferation and IL-2 and IFN-γ production were depressed in infected-castrated mice of both genders, while treatment with testosterone or dihydrotestosterone produced a significant cell proliferation recovery and enhanced production of IL-2 and IFN-γ (Morales-Montor et al., 2002a). An opposite effect of the same sex steroids was found on the humoral response: it was unaffected with testosterone or dihydrotestosterone restitution, while the treatment with estradiol in both genders augmented the levels of anti-cysticerci IgG, as well as IL-6 and IL-10 production. These results suggest androgens mediate immune functions, which protect mice from cysticercosis, possibly through the stimulation of the specific cellular immunity of the host

Immunoendocrine interactions during cysticercosis are the cornerstone of the feminization of male mice. When the infected male mice have an intact immune system, there is an increase in serum estradiol levels and a decrease of those of testosterone and DHT. However, when the immune system is knocked down by total irradiation or neonatal thymectomy, there is no change in the levels of serum steroids in chronically infected male mice, and the levels remain

The importance of sex-hormones driving the specific immune response during cysticercosis was assessed by administration of Fadrozole (a P450-aromatase inhibitor) in male and female mice to suppress the production of 17β-estradiol (Morales-Montor et al., 2002b). A reduction was found in parasite loads (~70%) in infected mice treated with Fadrozole. The protective effect of the P450-aromatase inhibitor was associated in male mice with a recovery of the specific cellular immune response. Furthermore, it has also been demonstrated that administration of Tamoxifen (an anti-estrogen) produced an 80% parasite load reduction in female mice, and had a weaker effect of 50% in male mice. This protective effect was associated in both sexes with an increase in the mRNA levels of IL-2 (a cytokine associated to protection against cysticerci) and IL-4 (innocuos against infection). Tamoxifen treatment modified 17β-estradiol production in females, while serum testosterone was not affected. However, the expression of the two types of estrogen receptor, ER-α and ER-β, in the spleen of infected mice of both sexes, was decreased by Tamoxifen treatment (Vargas-Villavicencio et al., 2007). The *in vitro* treatment of *T. crassiceps* with Tamoxifen, reduced reproduction and induced loss of motility in the parasite. These results indicate that Tamoxifen treatment is a new therapeutic possibility to treat cysticercosis, since it can act at both senses of the host-parasite relationship: increasing the cellular immune response protective against the parasite and acting directly upon the parasite, reducing its

steady between infected and uninfected male mice (Morales-Montor et al., 2001).

infection to a more parasite's permissive female environment.

(Morales-Montor et al., 2002a).

by the DNA homology between *T. crassiceps and T. solium* (Rishi & McManus, 1988). These characteristics have made murine cysticercosis a convenient instrument to test vaccine candidates and new drugs or treatments against cysticercosis (Vargas-Villavicencio). Several features of natural cysticercotic disease have been found by extrapolation from experimental murine cysticercosis (Morales-Montor et al., 2008).

#### **4.4 Role of sex steroids in cysticercosis**

In *T. crassiceps* cysticercosis, females of all strains of mice studied sustain larger intensities of infection than males, but during chronic infection (more than 4 weeks) this difference disappears and the males of BALB/c strain show a feminization process, characterized by high serum estrogens levels (200 times the normal values) whilst those of testosterone are 90% decreased. The target organs for testosterone action, testes and seminal vesicles, have a 50% weight reduction (Larralde et al., 1995). At the same time, the cellular immune response (Th1) is markedly diminished in both sexes, and the humoral (Th2) response is enhanced (Terrazas et al., 1998). Estradiol is involved in the immunoendocrine regulation of murine *T. crassiceps* cysticercosis as a major protagonist in promoting cysticercus growth interfering with the thymus dependent cellular immune mechanisms that obstruct parasite growth (Terrazas et al., 1994). Gonadectomy alters this resistance pattern and makes intensities equal in both sexes by increasing that of males and diminishing it in females (Huerta et al., 1992), whilst the serum sex steroids level are not detectable in these animals. However, the absence of estrogens does not prevent parasite growth in both genders, demonstrating that although estradiol favours *Taenia crassiceps* development, it is not indispensable for rapid parasite growth (Larralde et al., 1995).
