**5. Immuno-endocrine interactions in the host**

The mechanisms by which sex steroids act upon *T. crassiceps* cysticercus asexual reproduction are two: A) through the host's immune system mediation or/and B) directly through the parasites own physiological systems.

The changes in steroid production of infected male mice were found to associate with an increase in *c-fos* mRNA content in all tissues studied, whereas the *c-jun* mRNA content was increased only in the thymus. The p53 mRNA content was markedly reduced in all tissues of the parasitized animals analyzed, whereas bcl-2 gene expression was abolished only in the thymus (Morales-Montor et al., 1998).

On the other hand, thymic cell analysis performed by flow cytometry showed a diminution in the percent of CD3+CD4+, and CD3+CD8+ subpopulations in the infected mice, suggesting that the increase in estradiol levels of the host could change the expression pattern of several genes that participate in apoptosis regulation in the thymus of male mice during chronic infection with *T. crassiceps* cysticerci, and that estrogens could inhibit the specific cellular immune response to the parasite (Morales-Montor et al., 1998). Previous immunological experiments had led to suspect that estradiol positively regulates parasite reproduction in hosts of both sexes, presumably by interfering with the thymus-dependent cellular immune mechanisms that obstruct parasite growth (Th1) and favoring those that facilitate it (Th2) (Terrazas et al., 1998; Bojalil et al., 1993). A specific shift from Th1 to Th2 immune response in the course of infection was found that coincided with the initial low rate of reproduction that accelerates at later times of infection. The shift is characterized by a marked decrease of

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

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

The mechanisms by which sex steroids act upon *T. crassiceps* cysticercus asexual reproduction are two: A) through the host's immune system mediation or/and B) directly

The changes in steroid production of infected male mice were found to associate with an increase in *c-fos* mRNA content in all tissues studied, whereas the *c-jun* mRNA content was increased only in the thymus. The p53 mRNA content was markedly reduced in all tissues of the parasitized animals analyzed, whereas bcl-2 gene expression was abolished only in

On the other hand, thymic cell analysis performed by flow cytometry showed a diminution in the percent of CD3+CD4+, and CD3+CD8+ subpopulations in the infected mice, suggesting that the increase in estradiol levels of the host could change the expression pattern of several genes that participate in apoptosis regulation in the thymus of male mice during chronic infection with *T. crassiceps* cysticerci, and that estrogens could inhibit the specific cellular immune response to the parasite (Morales-Montor et al., 1998). Previous immunological experiments had led to suspect that estradiol positively regulates parasite reproduction in hosts of both sexes, presumably by interfering with the thymus-dependent cellular immune mechanisms that obstruct parasite growth (Th1) and favoring those that facilitate it (Th2) (Terrazas et al., 1998; Bojalil et al., 1993). A specific shift from Th1 to Th2 immune response in the course of infection was found that coincided with the initial low rate of reproduction that accelerates at later times of infection. The shift is characterized by a marked decrease of

murine cysticercosis (Morales-Montor et al., 2008).

**4.4 Role of sex steroids in cysticercosis** 

parasite growth (Larralde et al., 1995).

**5. Immuno-endocrine interactions in the host** 

through the parasites own physiological systems.

the thymus (Morales-Montor et al., 1998).

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 infection to a more parasite's permissive female environment.

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

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

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

The Role of Sex Steroids in the Host-Parasite Interaction 283

The evidence presented above illustrates the complexity and importance of neuroimmunoendocrine interactions during helminth infections, and provides clues to the many other possible mechanisms of parasite establishment, growth and reproduction in an immunocompetent host. Further, strong neuroimmunoendocrine interactions may have implications in the control of transmission and treatment of several parasitic diseases, but particularly in those produced by helminth parasites, in animals and humans. In practical importance, the complexity of the helminth-host relationship suggests that all physiological factors (i.e., sex, age) should be taken into account in the design of vaccines and new drugs. The differential response of helminthes to sex steroids may also be involved in their ability to grow faster in female or male hosts. Host and parasite sex-associated biases may be combined to favour their evolution towards a mutually acceptable relationship. Also, the strong immune-endocrine interactions observed during *Taenia crassiceps* and *Taenia solium* cysticercosis, could give ways to possible new mechanisms of parasite establishment,

Financial support was provided by grant IN 214011-3 from the Programa de Apoyo a Proyectos de Innovación Tecnológica, Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México to J. Morales-Montor. R.

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Barrabes, A.; Duong, TH. & Combescot, C. (1979). Effect of testosterone or progesterone

Benten, W.P.; Lieberherr, M.; Giese, G.; Wrehlke, C.; Stamm, O.; Sekeris, C. E.; Mossmann,

Besedovsky, H.O. & del Rey, A. (2002). Introduction: immune-neuroendocrine network.

Bojalil, R.; Terrazas, L.I.; Govezensky, T.; Sciutto, E. & Larralde, C. (1993). Thymus-related

cysticercosis (*Taenia crassiceps*). *J Parasitol*, Vol.78, pp. 471-476.

in breast and prostate cancer cells: targeting the association of Src with steroid

implants on the intensity of experimental infestation with *Schistosoma mansoni* in the female golden hamster. *C R Seances Soc Biol Fil*, Vol.173, No.1, pp. 153-156. Bebo, B.F.; Fyfe-Johnson, A.; Adlard, K.; Beam, A. G.; Vandenbark, A.A. & Offner, H. (2001).

Low-dose estrogen therapy ameliorates experimental autoimmune encephalomyelitis in two different inbred mouse strains. *J Immunol*, Vol.166, No.3,

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Hernández-Bello has a Posdoctoral fellowship from RED-Farmed, from CONACyT.

**7. Concluding remarks** 

**8. Acknowledgments** 

pp. 2080-2089.

**9. References** 

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reproduction and increasing its mortality (Vargas-Villavicencio et al., 2007). Another steroid that was recently tested and found to be implicated in the regulation of the parasite loads during murine cysticercosis is progesterone (P4). P4 treatment has a dichotomic effect: if mice of both sexes are non-gonadectomized (intact), P4 treatment increased parasite loads, possibly through manipulation of the specific cellular immune response, besides the steroid's promotion of parasite reproduction (Vargas-Villavicencio et al., 2005). However, if mice are gonadectomized, P4 completely decreases parasite loads, an impressive and unprecedented cysticidal effect, the likes of which are absent from other preventive or therapeutic measures (Vargas-Villavicencio et al., 2006). These two experiments suggests that, in intact hosts, progesterone is metabolized to estradiol, that is permissive for parasite reproduction, while in castrated animals, there is an active metabolism of progesterone in the adrenal glands to androgens, resulting in a toxic effect in the parasite growth (Vargas-Villavicencio et al., 2005, 2006). The major steroid produced by the adrenal gland is the androgen dehydroepiandrosterone (DHEA). So, another set of experiments showed DHEA effect on male and female infected mice. DHEA treatment reduced parasite loads by 70 and 80% respectively. In contrast with the common assumption of DHEA as an immunostimulatory hormone, the immune responses of our mice was not affected by DHEA treatment (Vargas-Villavicencio et al., 2008). *In vitro*, treatment of *T. crassiceps* cysticerci with DHEA induced an 80% reduction in parasite reproduction, which may partially explain the reduction of parasite loads observed *in vivo* a partial effect suggesting the involvement of other unknown factors in the *in vivo* regulation of parasite loads (Vargas-Villavicencio et al., 2008).

#### **6. Sexual steroids directly act upon** *Taenia crassiceps*

Not only do sex steroids regulate parasite loads through the immune response modulation of the host, but also they directly act upon cysticerci reproduction. For instance, it has been shown that E2 and P4 *in vitro* treatment stimulates *T. crassiceps* reproduction, while *in vitro* treatment with T or DHT inhibit and even exert a slight toxic effect on the parasite (Escobedo et al., 2004). The possible molecular mechanisms by which sex-steroids affect *Taenia crassiceps* reproduction, imply the presence of estrogen receptors (both α and β isoforms) and androgen receptors, but no progesterone receptors. In addition, once host's E2 has bound to its parasite estrogen receptor, the active, ligand bound complex would activate the transcription of several *Taenia crassiceps* proliferative genes, such as *c-fos, c-jun* and cyclin D1, and in that way up-regulate parasite growth and reproduction. All this hypothetical molecular mechanism could be interrupted *in vitro* by means of using tamoxifen that is well known for its anti-estrogenic effects (Vargas-Villavicencio et al., 2007), which strongly suggests a genomic action mechanism for 17-β estradiol on the parasite. On the other hand, action mechanism of the androgen is likely different from the found for estrogens and progesterone. Testosterone and DHT likely directly affect parasitic DNA integrity by activating apoptotic mechanism in the cysticercus cells. This experimental finding is not dependent of a nuclear receptor, because flutamide (a well studied and used anti-androgen) did not have effects upon parasite reproduction *in vitro* (Escobedo et al., 2004). These results demonstrate that sex steroids act directly upon parasite reproduction perhaps by binding to receptors closely resembling classic and specific sex-steroid vertebrate receptors (Escobedo et al., 2004) (64).
