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

Steroids 76 (2011) 1149–1159

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor Elizabeth Guadalupe Ibarra-Coronado a , Galileo Escobedo b , Karen Nava-Castro c , Chávez-Rios Jesús Ramses a , Romel Hernández-Bello a , Martìn García-Varela d , Javier R. Ambrosio e , ˜ f , Guadalupe Ortega-Pierres f , Lenin Pavón g , Olivia Reynoso-Ducoing e , Rocío Fonseca-Linán María Eugenia Hernández g , Jorge Morales-Montor a,∗ a

Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP 70228, México D.F. 04510, México Unidad de Medicina Experimental, Hospital General de México, México D.F. 06726, México c Departamento de Infectología e Inmunología, Instituto Nacional de Perinatología, México D.F. 11000, México d Departamento de Zoología, Instituto de Biología de la Universidad Nacional Autónoma de México, México D.F. 04510, México e Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Edificio A, 2do piso, Ciudad Universitaria, México D.F. 04510, México f Departamento de Genética y Biología Molecular, Cinvestav IPN, Av. Instituto Politécnico, Nacional 2508 Col. San Pedro Zacatenco 07360, Mexico g Departamento de Psicoinmunología, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, México D.F., México b

a r t i c l e

i n f o

Article history: Received 25 January 2011 Received in revised form 7 April 2011 Accepted 10 May 2011 Available online 19 May 2011 Keywords: Nuclear estrogen receptor Estradiol Taenia crassiceps Cysticercus Parasite Hormone receptors

a b s t r a c t 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-␤-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. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The sex steroid hormone 17-␤-estradiol (E2 ) acts upon the reproductive system of mammalians by binding to specific estrogen receptors (ER), which determines changes in reproductive physiology and behaviour [1,2]. Estrogens also transiently induce a number of nuclear proto-oncogenes, such as the c-fos and c-jun

∗ Corresponding author. Tel.: +52 55 56223158; fax: +52 55 56223369. E-mail addresses: [email protected], [email protected] (J. Morales-Montor). 0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2011.05.003

family proteins, which act as transcription factors through the ER system in the endometrial epithelium of mature and immature rodents. Changes in concentrations of these gene products presumably trigger the proliferation and differentiation of the uterine epithelium and mediate effects in areas of the brain under hormonal control [3,4]. Recently, estrogens, and particularly 17-␤estradiol, have been shown to participate not only in reproductive physiology, but in a number of different functions, including immune modulation, brain activity, bone metabolism, lung and heart physiology. Also, sex steroids influence a wide array of functions related to reproduction as well as to non-reproductive behaviours. The broad distribution, age, sex and tissue-depending

1150

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

expression pattern of estrogen receptors, as well as the functional disruptions in receptor knockout animals are solid evidence of the great diversity of the complex estrogen–estrogen receptor actions. ER operates as a hormone-induced transcription factor which prompts sexual receptivity in rats, hamsters, guinea pigs and mice [5]. The ER may be expressed either as ER-␣ or ER-␤ [6–8]; these variants are located in two different genes, and also differ functionally depending on the tissue in which they are expressed [6,9,10]. Furthermore, estrogens affect the pattern of immune response against different pathogens including parasites [11]. Recent experimental evidence suggests that steroid hormones have direct effects on different stages of both helminthes, Taenia crassiceps and Taenia solium [12–15]. Specifically, androgens reduce the reproduction and viability of T. crassiceps metacestodes in a concentration-dependent manner [12,13] while 17-␤-estradiol and progesterone stimulate the proliferation rate in this parasite [12]. Interestingly, tamoxifen (an anti-estrogen widely used in the treatment of estradiol-dependent breast cancers) exerts a strong toxic effect upon T. crassiceps, decreasing parasite reproduction in vitro and parasite loads in vivo [16]. Also, it has been shown that progesterone increases T. solium scolex evagination and worm growth in a concentration-independent way, while RU486, a progesterone antagonist, inhibits either scolex evagination or worm development induced by progesterone [14]. Despite of the fact that there is a clear direct effect of steroid hormones on some parasites, the mechanisms involved have not been fully defined yet. Onchocerca volvulus has a nuclear receptor potentially able to bind retinoic acid [17]. Furthermore, sequences related to a progesterone receptor were detected by RT-PCR and Western blot in the helminth parasite T. solium [14]. Moreover, the presence of a possible mRNA sequence similar to an estrogen receptor has been shown in T. crassiceps cysticerci [12] by RT-PCR and by sequencing a specific amplified fragment. The present study was designed to search for an estrogen receptor-like molecule, which could mediate the proliferative effects of exogenous and endogenous 17-␤-estradiol on the helminth parasite T. crassiceps. Results showed that T. crassiceps indeed has a protein similar in function to an ER, which plays a role in parasite development. These findings may improve our understanding of the host-parasite molecular cross-talk, and could also represent a target for the design of new drugs specifically directed to arrest the activity of key parasite molecules, such as transduction proteins and transcription factors involved in their establishment, growth and reproduction inside an immunocompetent host.

of in vitro treatment of estradiol and tamoxifen on cysticerci. Cultures were checked every day and their medium was completely replaced each 24 h or when it turned yellowish. 2.2. Ethics statement Animal care and experimentation practices at the Instituto de Investigaciones Biomédicas are frequently evaluated by the Institute’s Animal Care and Use Committee, and by governmental agencies, in strict accordance with the recommendations set forth in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the USA, to ensure compliance with established international regulations and guidelines. The protocol was approved by the Ethics Committee for Animal Experiments of the Instituto de Investigaciones Biomédicas (Permit Number: 2009-16). Mice sacrifice to obtain control tissues was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. 2.3. In vitro treatment effects of E2 and tamoxifen on T. crassiceps cysticerci reproduction Culture grade 17-␤-estradiol (E2 ) and tamoxifen were obtained from Sigma. For in vitro tests, water-soluble E2 was dissolved in DMEM serum-free culture medium, while tamoxifen was dissolved in absolute ethanol. Next they were each prepared at a concentration of 10 mg/ml and then sterilized by passage through 0.2 mm Millipore filter paper. Each of the following experimental conditions was applied to 24 parasite-loaded wells to obtain the concentration-response curves: (a) parasites were supplemented with serum-free DMEM as vehicle (control groups), (b) parasites were separately supplemented with 5, 10, 20 and 40 ␮g/ml of E2 and (c) parasites were supplemented with 1, 10, 15, 40 and 80 ␮g of tamoxifen. Optimal concentrations of E2 and tamoxifen were selected from concentration-response curves, and later used for time-response curves. Final concentrations were: 40 ␮g/ml medium of E2 and 40 ␮g/ml medium of tamoxifen. The number of buds per cysticercus as a function of days elapsed in culture was assessed as the variable response. Tamoxifen was supplemented 2 h before the addition of 17-␤-estradiol. Parasite reproduction was measured by counting the total number of buds in the 10 cysticerci in each well. Bud count, as well as viability, was checked every day under an inverted light microscope (Olympus, MO21, Tokyo, Japan) at 4× and 10× magnification. Injury to cysticerci was recognized microscopically by progressive internal disorganization, development of whitish opaque areas on the parasite’s tegument and loss of motility. Dead cysticerci were immobile, opaque and structurally disorganized.

2. Experimental 2.1. Parasites

2.4. Detection of ER-like gene expression in T. crassiceps by RT-PCR

Cysticerci were obtained from intraperitoneally infected mice and placed in tubes containing sterile PBS (1X) supplemented with 100 U/ml of antibiotics-fungizone (Gibco, Grand Island). The tubes were centrifuged for 10 min at 200 × g at 4 ◦ C, and the supernatant was discarded. Packed cysticerci were incubated in DMEM serumfree medium (Gibco 12491). The parasites were then centrifuged 3 times at 200 × g for washing. After the final wash, viable cysticerci (complete, translucent and motile cystic structures) were counted under a stereoscopic microscope. Ten viable non-budding cysticerci of approximately 2 mm in diameter were then selected and dispensed into each well of 24-well culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, New Jersey) in 1 ml DMEM medium (Gibco 12491) and incubated at 37 ◦ C and 5% CO2 . A sufficient number of culture wells was prepared to evaluate the effects

Total RNA was isolated from untreated, E2 - and tamoxifentreated T. crassiceps cysticerci, and from BALB/c AnN female mouse uterus as control for specific ER gene amplification by the single-step method based on guanidine isothiocyanate/phenol/chloroform extraction using Trizol reagent (Invitrogen, Carlsbad, CA). Briefly, cysticerci were disrupted in Trizol (1 ml/0.1 g tissue), and 0.2 ml chloroform were added per 1 ml of Trizol. The aqueous phase was recovered after 15 min centrifugation at 16,000 × g. RNA was precipitated with isopropyl alcohol, washed with 75% ethanol and dissolved in RNAse-free water. RNA concentration was determined by absorbance at 260 nm and its purity was verified by electrophoresis in 1.0% denaturing agarose gel in the presence of 2.2 M formaldehyde. Total RNA was reverse-transcribed followed by specific PCR amplification of

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

the ER-like gene from parasite tissue. The ribosomal 18S gene was used as control, as described elsewhere [18]. Briefly, 10 ␮g of total RNA were incubated at 37 ◦ C with 40 units of M-MLV reverse transcriptase (Applied Biosystems, USA) in 20 ␮l of reaction volume containing 50 ␮M of each dNTP and 0.05 ␮g oligo (dT) primer (Gibco, Invitrogen, NY). Ten microlitres of the cDNA reaction were subjected to PCR in order to amplify the sequences of the specific genes. Primer design was based on the most conserved regions of sequenced genes of all species reported in the database. Sequences of primers were as follows: ER-␣ (239 bp) sense 5 -AGACTGTCCAGCAGTAACGAG-3 and the antisense primer sequence 5 -TCGTAACACTTGCGCAGCCG-3 . The 50 ␮l PCR reaction included 10 ␮l of previously synthesized cDNA, 5 ␮l of 10× PCRbuffer (Perkin-Elmer, USA), 1 mM MgCl, 0.2 mM of each dNTP, 0.05 ␮M of each primer, and 2.5 units of Taq DNA (Biotecnologias Universitarias, Mexico). After an initial denaturing step at 95 ◦ C for 5 min, temperature cycling was as follows: 95 ◦ C for 30 s, 57 ◦ C for 45 s and 72 ◦ C for 45 s during 35 cycles. A further extension step was completed at 72 ◦ C for 10 min for each gene. The 20 ␮l of the PCR reaction were electrophoresed on 2% agarose gel in the presence of a 100 bp ladder as molecular weight marker (Gibco, BRL, NY). The PCR products obtained were visualized by staining with ethidium bromide. In both cases, different PCR conditions were assessed until a single band corresponding to the expected molecular weight of the gene was found. The ribosomal 18S gene is a constitutively expressed gene and it was used as internal loading control. The sequence of the house-keeping gene was as follows: 18S (238 bp) sense primer 5 -GGGTCAGAAGGATTCCTATG-3 , and antisense primer 5 -GGTCTCAAACATGATCTGGG-3 . 2.5. ER-like protein detection in T. crassiceps by Western blot Total protein was obtained from T. crassiceps cysticerci by conventional Tris–HCl isolation. Briefly, non-treated, E2 and tamoxifen-treated cysticerci were disrupted in Tris–HCl (1 ml/0.1 g tissue) and proteases inhibitor cocktail (Calbiochem). Mouse uterus was used as internal control of protein extraction and integrity. The supernatant was recovered after 15 min centrifugation at 16,000 × g and the pellet was discarded. Protein quantity was determined by absorbance at 595 nm using the Bradford-Lowry method. Thirty micrograms of total protein extracts of T. crassiceps cysticerci and mouse uterus were boiled in reducing Laemmli sample buffer, separated by SDS-PAGE (10% acrylamide) and transferred onto PVDF membranes. The membranes were blocked overnight in PBS buffer (0.2% Tween 20) containing 1% BSA. Then, different membranes were washed 5 times in PBS-Tween and separately incubated for 1 h in presence of rabbit anti-ER-␣ (1:300, Santa Cruz Biotechnology). After this first incubation, membranes were washed 3 times in PBS-Tween and subsequently incubated for 1 h in presence of goat anti-rabbit IgG-HRP (1:500, Santa Cruz Biotechnology Amersham) as secondary antibody. Immediately after the bands were visualized the reaction was stopped using the ECL system according to the manufacturer’s instructions (Super Signal ECL, Pierce). Chemiluminescent signals were captured on Kodak BioMax film. 2.6. Immunoprecipitation and specific detection of ER-like protein in Taenia crassiceps

1151

Once the supernatant was discarded, the pellet was resuspended in 30 ␮l lysis buffer and 30 ␮l of Laemmli loading buffer. 2.7. Specific detection of ER-like protein in T. crassiceps cysticerci by flow cytometry T. crassiceps cells were extracted by tissue disruption from cultured treated and non-treated parasites. Mouse spleen cells were used as FACS calibration control. For each treatment, 2 × 106 cells were incubated at 4 ◦ C for 20 min in presence of anti-CD3 and antiMHC-II or anti-CD4, anti-CD8 and subsequently washed in sterile PBS 1X-staining. Next, cells were centrifuged at 300 × g for 5 min, and incubated in GolgiPlug for 3 h. Cells were then washed in Perm/Wash buffer and centrifuged at 11,000 × g for 5 min. After this, cells were separately incubated in presence of rabbit ␣-ER 1 ␮g/␮l (Santa Cruz, Biotech) and mouse ␣-paramyosin 1 ␮g/␮l at room temperature for 20 min, and subsequently washed in PBS 1Xstaining. After this step, cells were centrifuged at 300 × g for 5 min. Cell pellets were resuspended separately in presence of the secondary antibody FITC-conjugated goat anti-rabbit or PE-conjugated rat anti-mouse antibody, and incubated at 4 ◦ C for 30 min in the dark. After this second incubation, cells were washed in PBS 1Xstaining solution and centrifuged at 300 × g for 5 min. Cell pellets were resuspended in 500 ␮l of PBS 1X-staining solution in absence of light and analyzed by flow cytometry using a FACS Calibur (BD, Biosciences). Data were analyzed with the FlowJo software. 2.8. ER-like protein location on T. crassiceps cells by immunofluorescence Cultured T. crassiceps cysticerci were washed with PBS 1X, embedded in Tissue Tek (Triangle Biomedical Science), and frozen at −80 ◦ C. Parasite tissue sections (5 ␮m) were fixed with 4% paraformaldehyde for 30 min, washed 3 times in PBS and blocked for 30 min with RPMI medium containing 0.5% albumin bovine and 5% fetal bovine serum [19]. Cross-sections were then incubated with a 1:500 dilution of rabbit anti-ER-␣ (Santa Cruz, Biotech) for 45 min at 37 ◦ C, washed with PBS and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Zymed) at 1:200 dilution. Control experiments were assessed incubating the 5 ␮m thick tissue sections in presence of only the FITC-conjugated goat anti-rabbit antibody at the same dilution. To eliminate background fluorescence, samples were contrasted with 0.025% Evans Blue for 10 min. After two single washings, samples were mounted in Vectashield mounting medium (Vector Laboratories Inc.) and examined with a Carl Zeiss epifluorescence microscope at 10× and 40× magnification (Carl Zeiss, Germany). 2.9. Cell sorting of T. crassiceps cells For cell sorting, cells were obtained as previously stated, and were first selected by size and granularity into 4 subpopulations of parasitic cells, and electronically purified using a FACSAria cell sorter (Becton & Dickinson). Cells were recovered into an Eppendorf tube containing 5% of culture medium supplemented with bovine fetal serum. Cells were then used to perform binding experiments. Purity of the sorted populations was above 96%. 2.10. Preparation of cysticercus proteins

For specific detection of ER-like protein in T. crassiceps, 30 ␮g of total protein from the parasite were mixed with 1 ␮g of rabbit anti-ER-␣ during 1 h at 4 ◦ C. Then 30 ␮g of Protein G was added and the mixture was incubated overnight at 4 ◦ C. After centrifugation at 5,000 × g for 1 min, the conjugate was washed 3 times with 800 ␮l lysis buffer (Tris–HCl and proteases inhibitor cocktail) and centrifuged each time at 5000 × g for 1 min at room temperature.

After recovering the parasites, these were sonicated 3 times with a period of amplitude frequency of 50 kHz and 1 min intervals between each sonication. Protein purification was performed after its precipitation at 4 ◦ C using a −20 ◦ C frozen acetone solution containing 10% TCA/20 mM DTT. Precipitated proteins were resuspended in a commercial 5 mM Tris-Base complemented with

1152

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

inhibitors (pepsin 1 mg/ml, pepstatin 2 ␮g/ml, aprotinin 5 ␮g, leupeptin 5 ␮g/ml, PMSF 1 mM) or in 6.7 mM phosphate buffer adjusted to pH 7.4 with 0.04 M KCl and 1 mM MgCl2 in presence of proteases inhibitor. Resultant proteins were quantified using the DC Protein Assay commercial kit and bovine serum albumin as a standard. Protein aliquots were frozen at −70 ◦ C until used. 2.11. Electrophoresis Ten percent SDS-PAGE gels were run for protein stacking at 80 V during 20 min, and electrophoretic conditions were 100 V during 90 min. After separation, proteins were revealed by Coomassie Blue staining (PhastGel Blue R). The standard commercial molecular weight marker was kaleidoscope (Bio-Rad). Gels were unstained using a solution containing glacial acetic acid, methanol and water. Gel images were captured with an XRS ChemiDoc (Bio-Rad) photodocumentation device equipped with Quantity One software Vdkdkd. 2.12. Isoelectrofocusing IPG strips (GE) of 7 cm length (pH 3–10) were hydrated at room temperature for 14 h in the presence of protein (50 ␮g) and a rehydratation solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 3–10 2% ampholytes, 60 mM DTT and 0.002% bromophenol blue. Proteins were separated by pI in a Protean IEF Cell equipment (Bio-Rad) at 20 ◦ C using 50 ␮A/strip in a discontinuous current as follows: step 1 with a linear slope during 20 min at 250 V, step 2 with a linear slope during 2 h at 4000 V and step 3 with a rapid steep slope beginning at 4000 V and ending at 10,000 V. After finishing, strips were recovered and frozen at −70 ◦ C until used. 2.13. Second dimension electrophoresis After isoelectrofocusing, strips were molded with 0.5% hot agarose and 2D electrophoresis was performed at 4 ◦ C using a commercial system (NuPAGE® Bis–Tris electrophoresis system) with pre-cast 4–12% mini gels at 200 V during 40 min. During runnings, NuPAGE® antioxidant reagent for reducing conditions was used. All reagents were from Invitrogen. Gels were stained with Sypro-Ruby (GE, Healthcare) or Blue Coomassie following the suppliers’ indications. Other 2D gels were processed for protein electrotransfer to PVDF membranes as indicated below. Gel images were captured with the PDQuest software (BioRad). Exact pI values were calculated after printing the images and tracing the MW vs. pI coordinates, according with standard MW markers. The apparent MW/pI parameters of each resultant spot was estimated. Selected spots were cut out from gels in 18.2 m MilliQ water and maintained at 4 ◦ C until further processing. 2.14. Protein electrotransference Proteins on SDS gels were transferred, under reducing conditions, to PVDF membranes (GE, Healthcare) using a commercial equipment XCell II Blot Module coupled to a chamber XCell SureLockTM Electrophoresis Cell and Novex Mini-Cell in presence of buffer (NuPAGE® Transfer Buffer). All reagents were obtained from Invitrogen. 2.15. ER-like protein detection by 2D electrophoresis and Western blot Briefly, after processing proteins and separating them by 2D gel electrophoresis, they were transferred into nitrocellulose membranes and Western blot was performed using an anti-ER-␣ antibody, as mentioned before. Only the immunodetected point,

corresponding to the expected molecular weight and to the predicted isoelectrical point is highlighted in the blotted membrane. 2.16. Phylogenetic analysis of the putative T. crassiceps estrogen binding protein The protein sequence of the T. crassiceps estrogen binding protein was aligned to the ER-␣ protein sequences of other 13 species (including mammals, birds, fish, one reptilian and one amphibian) obtained from protein data sets in GenBank. Sequence alignment was done using Clustal W software. Alignment of contained 131 amino acids (aa) from 13 different taxa. Phylogenetic relationships were inferred using the Neighbor joining (NJ) method. Robustness of the NJ tree was evaluated using bootstrap of 10,000 replicates. The tree was drawn using RETREE and DRAWGRAM from PHYLIP. The genetic differentiation between taxa was estimated using the mean character difference with the help of PAUP* 4.0b10 software. It is important to point out that the number of species used for the analysis was selected based on the ER-␣ sequence found in the Gene Data Bank (for some species there is only one sequence). 2.17. Experimental design and statistical analysis E2 and tamoxifen concentration-response and time-response curves were estimated from six independent experiments; each was performed with 10 cysticerci, freshly extracted from different infected donor female mice. Each experiment was replicated in 24 different wells. The response variable used in statistical analysis was the sum of buds present in the 24 wells with each treatment and time of exposure of the experiments. Data from the six replications of each experiment were pooled and expressed as mean ± SD. All optical densitometries as well as mean fluorescence of the flow cytometry analysis were calculated for 4 different experiments, and expressed as mean ± SD. Data were analyzed using either Student’s t-test or one-way ANOVA and subsequently with Dunnet’s Multiple Comparison Test, depending on the experimental design. Differences were considered statistically significant with P < 0.05. 3. Results 3.1. E2 stimulates while tamoxifen diminishes T. crassiceps reproduction Bud number of cultured T. crassiceps cysticerci clearly increased upon addition of 17-␤-estradiol, in a concentration-dependent manner. Compared to control groups, E2 increased parasite reproduction rate 2-fold at the lowest concentration (0.1 ␮g/ml), and more than 10-fold at the highest concentration (40 ␮g/ml), without affecting parasite viability (Table 1). In addition to the concentration effects, the proliferative action of E2 on parasite reproduction was maintained throughout the approximately five days of in vitro culture (Table 1). In contrast, tamoxifen showed the opposite effect on bud number, although this effect was only significant with the highest drug concentrations (20 and 40 ␮g/ml). This suggests that the tamoxifen effect was also dependent on drug concentration (Table 2). Interestingly, when compared with control cysticerci, tamoxifen treatment of the larvae had no significant effects on parasite basal reproductive rate (Table 2). However, it is clear that tamoxifen completely blocked the E2 dependent proliferative effect (Table 2), emphasizing the importance of the E2 -dependent proliferative mechanisms in T. crassiceps cysticercus reproduction. 3.2. ER-like mRNA gene expression in Taenia crassiceps Using specific primers, which were designed considering the most conserved sequences of all ER reported to date, we were able

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

1153

Table 1 Dose-response curve reproduction of cultured Taenia crassiceps cysticerci in response to estradiol. Estradiol concentration (␮g/ml)

Total number of buds

0 0.1 1 10 20 40

10 24 36 73 96 123

± ± ± ± ± ±

1.123 9.8944 11.0954 20.7527 23.8164 25.7527

Cysticerci motility *** **** **** ***** ***** *****

Data represent mean ± SD number of buds from 3 experiments, with 10 cysticerci per well, 6 wells per dose, during five days of culture. * Refers to a scale used by us to describe the motility of the organism. Injury to cysticerci was recognized microscopically by progressive internal disorganization, development of whitish opaque areas on the parasite’s tegument, and by loss of motility. Dead cysticerci were immobile, opaque, and disorganized structures. (***) 50% of cysticerci motile, but 100% alive, (****) 80% of cysticerci motile, but 100% alive, (*****) very motile, 100% alive and healthy in their appearance.

Table 2 Dose-response curve of tamoxifen effect on estradiol-stimulated reproduction of cultured T. crassiceps cysticerci. Tamoxifen (␮g/ml)/estradiol (␮g/ml) concentration 0/0 0/1 5/10 10/15 20/40 40/80

Total number of buds 10 24 16 13 6 3

± ± ± ± ± ±

1.123 0.8944 1.0954 0.7527 0.8164 0.7527

Cysticerci motility ***** ***** **** *** ** *

Data represent mean ± SD from 3 experiments, with 10 cysticerci per well, 6 wells per dose. * Refers to a scale used by us to describe the motility of the organism. Injury to cysticerci was recognized microscopically by progressive internal disorganization, development of whitish opaque areas on the parasite’s tegument, and by loss of motility. Dead cysticerci were immobile, opaque, and disorganized structures. (*****) Very motile, and healthy in their appearance, (****) less than 80% of cysticerci motile, but still 100% alive, (***) less than 50% of cysticerci motile, but still 100% alive, (**) no motility in 80% of the cysts, with a reduction of 50% in survival, (*) no motility in 100% of the cysts, with 100% mortality reached.

to amplify a band of 239 bp corresponding to the expected length of the T. crassiceps ER-like gene (Fig. 1A) and to the ER gene of mouse uterus used as internal control. Interestingly, no difference in ERlike gene expression was observed between E2 -treated and control parasites. However, tamoxifen-treated cysticerci showed a significant down-regulation of T. crassiceps ER-like gene expression, even in presence of E2 (Fig. 1B). 3.3. ER-like protein expression by Western blot and immunoprecipitation in T. crassiceps

Fig. 1. Expression of an estrogen receptor-like protein in Taenia crassiceps. (A) A single band of approximately 239 bp corresponding to ER was detected in all in vitro 17-␤-estradiol-treated cysticerci. Tamoxifen treatment significantly inhibited the ER -like protein expression compared to control cysticerci. (B) Relative expression of the T. crassiceps ER-like gene using 18S rRNA as constitutive gene. E2 , cysticerci treated with 17-␤-estradiol; Tmx, cysticerci treated with tamoxifen; Tmx + E2 , cysticerci treated with tamoxifen plus 17-␤-estradiol; C, cysticercus culture without treatment; CEtOH , cysticercus culture with vehicle; uterus, mouse uterus. * P < 0.05, ** P < 0.01.

CD4+ and CD8+ which are typically present in some types of mammalian leukocytes, but only expressed paramyosin (Ag-B), an exclusive cytoskeleton component of cestodes, nematodes and some insects (Fig. 4A) [20–24]. This parasite cell population was selected for further ER-like expression analyses. In contrast with spleen cells, which were CD3+ /MHC class II+ (Fig. 4B), the ERlike protein was mainly recognized on T. crassiceps cells, which were CD3− /MHC class II− (Fig. 4C–E). Interestingly, the fluorescence intensity related to the ER-like protein observed in E2 -treated parasites was 3-fold above the level observed in untreated control cysticerci (Fig. 5). 3.5. ER-like protein is present in the sub-tegumental cells of T. crassiceps cysticerci

Native ER-like protein was detected in untreated, and in E2 and tamoxifen-treated parasites by the Western-blot method. The ER-like protein of T. crassiceps was detected, in addition to some unspecific bands (Fig. 2A). Immunoprecipitation was then used to obtain the specific ER-like protein from the parasite. Contrary to the data obtained by RT-PCR, no changes in total protein quantity were observed with any treatment and the ER-like protein was detected in all treatments (Fig. 2B).

As mentioned above, experimental evidence indicated that ERlike protein is expressed in T. crassiceps cysticerci, and is not a contamination product from host cells. Thus, immunofluorescent assays on well-preserved T. crassiceps tissue were performed, and results showed that parasite cells express ER-like protein mainly in the subtegumental cysticercus tissue (Fig. 6C and D). As expected, T. crassiceps tissue incubated only in the presence of secondary antibody gave no positive signal (Fig. 6B), which demonstrated that the experimental conditions were optimal for detecting exclusively parasite cells presenting ER-like molecules and not false positive signals.

3.4. ER-like protein is specifically detected in T. crassiceps and it is not a contamination product from host immune cells

3.6. Identification of the T. crassiceps ER-like protein by 2DE blotting

Flow cytometry analysis first showed that T. crassiceps cells differed in size and complexity from mouse spleen cells. In fact, parasite cells were approximately 3-fold smaller and showed less complexity (Fig. 3A) than mouse spleen cells (Fig. 3B). In addition, parasite cells showed no expression of the membrane markers

After bidimensional electrophoresis of total proteins from E2 -treated parasites, the spots were resolved in the Coomassie Blue-stained gels (Fig. 7A). Most spots displayed neutral and alkaline pI with a wide range of MW, and some were well-recognized by polyclonal antibodies as shown in Fig. 7B. Since well-characterized

1154

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

Fig. 2. (A) Western blot of T. crassiceps ER-like protein and (B) specific immunoprecipitation of T. crassiceps ER-like protein. (A) ER-like protein from cultured cysticerci, and mouse uterus ER protein were purified from total protein extracts. (B) Immunoprecipitation assays showed that the ER-like protein expression level was similar in treated and non-treated cysticerci. Arrow indicates the ER protein.

estrogen receptors have been localized around pH values of 6.0 and MW values of 66 kDa, the possibility of this molecule being an ER was high. Based on antibody recognition, other ER-like isoforms could also perhaps be present in the same MW band of the identified protein. Sequencing of the spot yielded a small fragment of protein similar to a mammalian nuclear estrogen receptor, which corresponds to the translation product of the mRNA previously sequenced by us (Gene Data Bank accession number AY596184) (Fig. 7C). 3.7. Phylogenetic analyses and Sequence alignment A posterior analysis of the T. crassiceps protein showed homology of around 60% to the protein sequences previously reported for mouse, rat, rabbit and human ER-␤ in the Gene Data Bank. It is important to mention that the analyzed conserved motif was situated in a region of approximately 90 aa, located in the DNAbinding domain of the C-terminal motif (from aa position number 110 to 160 of the mammal sequences described above). A more precise analysis of the T. crassiceps putative ER-␤ sequence involved a Neighbor Joint Tree (NJT) for studying phylogenetic relationships (Fig. 8). The partial sequence of the estrogen-binding protein from T. crassiceps obtained in the present study was aligned to other 13 sequences, conforming a data set of 396 aa. This NJT was

inferred from the ER-␣ dataset, producing a single tree composed by 5 groups. The maximum parsimony tree inferred with this data set, yielded single tree with a length = 701, and with a consistence index = 0.95. This unrooted tree (Fig. 8) was composed by 5 groups. The first contains sequences of 3 mammals (rat, human and natural host of the parasite) with strong bootstrap support of 100%. The second group consisted of one reptilian and one bird. The third group included two sequences of amphibians. The fourth group includes sequences of 3 species of fish. Finally the fifth group contains 3 sequences of Platyhelminthes, including T. crassiceps. The phylogenetic relationships among the 5 groups received good bootstrap support ranging from 66% to 100% (see Fig. 8). Additionally, T. crassiceps ER-␣ is related to the ER-␣ family of vertebrates, more closely associated to reptilian and amphibian (Fig. 8). This finding suggests that T. crassiceps ER-␣ is definitively not a product of host cell contamination, specifically not of mouse or human cells, because of the big distance between T. crassiceps and mammals in the NJT. 4. Discussion As we reported previously, 17-␤-estradiol exerts a direct proliferative effect on T. crassiceps cysticercus reproduction, which is not necessarily mediated by the host’s immune system but by a classic nuclear receptor in the parasite [12]. The aim of the present

Fig. 3. Comparison between complexity and size of T. crassiceps cells and mouse spleen cells. (A) Parasite cells show smaller size and similar complexity as spleen cells. (B) Characteristic mouse spleen showing normal size and complexity. SSC = Side scatter; FSC = Forward scatter. Results are representative of 3 experiments.

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

1155

Fig. 4. Specific detection of an ER-like protein in T. crassiceps. FACS analysis showed that the ER-like protein detected in T. crassiceps was not a contamination product from host immune CD3 and MCH class II positive cells. (A) Parasite cell paramyosin+ , CD4− /CD8− . (B) Mouse spleen cells CD3+ /MHC II+ . (C) Basal inmmunofluorescence in cysticerci cells with no added antibodies. (D) Untreated parasite ER-like protein+ , CD3− /MHC II− . (E) Parasite cells treated with estradiol (40 ␮g/ml) ER-like+ , CD3− /MHC II− . Controls = Parasites treated with the vehicle in which hormone and tamoxifen were dissolved. Results are representative of 3 experiments.

Fig. 5. ER-like protein expression in T. crassiceps cells. (A) Mean fluorescence intensity of ER-like protein expression in parasite cells. In non-treated cysticerci very few cells presented a low immnofluorescent signal (thin line) while estradiol-treated cysticerci showed few cells with a high immnoflurescent signal related to the ER-like protein expression level (thick line). (B) Graphic representation of mean fluorescence intensity. Estradiol-stimulated cysticerci showed a 3-fold increase in fluorescence intensity compared with control non-treated parasites. Results are representative of 3 experiments.

1156

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

Fig. 6. Localization of an ER-like protein in T. crassiceps by immunofluorescence. (A) Transversal section of one T. crassiceps cysticercus where tegument, sub-tegument and cells are observed with Nomarski. (B) Negative control of immunofluorescence using the secondary antibody. (C) Specific detection of ER-like protein (arrows) in parasite cells mainly located along subtegumental tissue, 10×. (D) A magnification of 40× from “C” (area boxed) shows details of the T. crassiceps cells expressing ER-like protein exclusively on subtegumental cells (arrows). T, tegumental cells; GL, germinal layer.

Fig. 7. ER-like detection by 2D electrophoretic analysis. (A) Total protein of in vitro cultured T. crassiceps was resolved in a 2D gel. (B) A well-defined dot of 66 kDa, pI = 6.0 was detected in T. crassiceps using an antibody to detect the protein (red circle). Molecular weight of proteins are as indicated in the figure. (C) Spot sequencing produced a small fragment of protein similar to a mammalian nuclear estrogen receptor (Gene Data Bank accession number AY596184). Arrows indicate protein isoelectrofocusing (pH 4–7) and their resolution in the second electrophoresis running using MW commercial standards as indicated. In A, the gel was stained by Coomassie Blue and in B, proteins were resolved in chemiluminescence assays after their recognition by primary and secondary antibodies.

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

1157

Fig. 8. Maximum parsimony tree inferred from a data set of 396 aa. Numbers near nodes show MP bootstrap frequencies. Neighbor Join Tree (NJT) for phylogenetic relation analysis. ERs from several species of fish, amphibian, reptilian, bird and mammals were analyzed through a NJT for searching probable relationship to the T. crassiceps ER identified and sequenced. The T. crassiceps protein showed close relation to ERs from fish and amphibian, but distant to their counterparts in mammals. Numbers on the NJT means bootstrap support ranging among analyzed species.

study was to investigate the participation of an estrogen receptorlike molecule in the helminth cestode T. crassiceps, with possible reactiveness to endogenous estradiol which could be involved in the parasite’s proliferative processes. In earlier work, we found that gender and circulating E2 levels in host mice crucially affect the dynamics of parasite loads in mice infected with T. crassiceps cysticerci [25,26]. Infection of male mice with T. crassiceps leads to striking increments in host estrogen levels, which is consistent with the notion that cysticerci reproduce better in high estrogen conditions and somehow induce its production in the host. An inadequate hormonal environment may lead to apoptosis of crucial parasite cells, as has been proposed for other parasite infections, i.e., retinoic acid has been shown to affect female Litomosoides carinii and microfilariae of L. carinii, Brugia malayi, Brugia pahangi, and Acanthocheilonema viteae [27]. Cercariae, schistosomula, and adult worms of Schistosoma mansoni show reduced viability and significant inhibition of in vitro schistosoma oviposition [28] while testosterone does likewise with the mitochondrial function of S. mansoni [29]. Because there is a great deal of conserved sequence homology among most hormone receptors, especially in the ligand and DNA-binding domains [30], we were able to show that cysticerci expresses an ER. Such similarities between host and cysticercus metabolism are not surprising since extensive homologies between species are being documented in other systems as well. Interestingly, present results show that only one isoform of the classic ER (ER-␣) is expressed in T. crassiceps. It appears that the binding of E2 to its respective receptor causes the effects of estrogens. Binding of the ER to the classic estrogen-

dependent elements could be responsible for the activation of AP-1 complex genes in the normal metabolism of T. crassiceps. Previous studies have demonstrated that the genome of O. volvulus encodes at least 3 members of the nuclear receptor family [31], and this could also be the case for T. crassiceps. The present study revealed that progressively higher 17-␤estradiol concentrations correlate with the number of T. crassiceps cysticercus buds. The opposite response was observed when a competitive inhibitor of estradiol-binding to the ER (tamoxifen) was tested in culture. As tamoxifen concentration increased, parasite reproduction progressively decreased. On the other hand, the effect of E2 was enhanced as time passed, reaching its maximum effect on parasite reproduction at day five of in vitro culture, which supports the fact that E2 effects depended on its concentration. Nevertheless, no time-dependent response was found when tamoxifen was tested, although complete blockage of the E2 dependent proliferative effect occurred since the first day suggesting that the parasite ER-like protein is directly involved in mediating the cross-talk between the hormonal microenvironment (exogenous E2 ) and parasite reproduction. These findings support previous results showing a marked concentration and time-dependent pattern regarding the effects of E2 on cysticercus reproduction. These observations are relevant since they suggest that sex-steroids may have similar effects on mammals and on parasitic cestodes, a hypothesis that evokes the wide range of effects of steroid hormones not only on several different cell types, but also along the phylogenetic scale, among distantly related organisms.

1158

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159

Moreover, determination of the effects of E2 and tamoxifen on T. crassiceps cysticerci allowed to determine the expression and translation of the ER-like gene into functional proteins which mediate the E2 effects. Based on this, a band corresponding to the ER-like gene was amplified from T. crassiceps larval tissue by means of specific primers designed considering the most conserved regions of this gene, which have been previously reported in mammals such as the mouse, rat and human, as well as in yeast, birds and amphibians. Furthermore, not only is the ER-like gene expressed in the parasite, but it can also be significantly downregulated by tamoxifen. This unexpected finding suggests that helminth parasites may have developed a positive feed-back mechanism able to sense changes in the expression of very important molecules (such as ER) and maintain their activity in order to avoid compromising the viability and reproduction of the parasite. Also, this finding offers an alternative explanation to the finding that T. crassiceps cysticerci grow better in female mice than in male mice [12], and emphasizes the molecular cross-talk between host and parasite, which, in turn, is differentially influenced by the hormonal microenvironment of each gender. A critical aspect of the present study was to determine that the detected and analyzed ER-like protein was exclusively found in the T. crassiceps cysticercus, and it was not a consequence of host immune cell contamination, because, as shown elsewhere, there is extremely high interaction between parasites and immune cells, which may eventually lead to leukocyte invasion of several parasitic tissues [32]. For this reason, an alternative use of flow cytometry analysis was used to differentiate proteins from T. crassiceps and the murine host by identifying molecules exclusive of the parasite that are neither synthesized nor expressed by the host. This is the case of paramyosin, a muscle protein found only in invertebrates such as Drosophila melanogaster, Caenorhabditis elegans, T. solium and T. saginata [33]. The flow cytometry studies showed the presence of a specific ER-like protein in the parasite, because paramyosin was only detected in T. crassiceps cells, where this estrogen receptor-like molecule was also found and studied. In contrast, the anti-paramyosin antibody did not recognize cells extracted from mouse spleen, which in addition were positive for CD4 and MHC class II antibodies, contrary to parasite cells. These results show that the origin of the analyzed ER-like protein was in fact T. crassiceps, and simultaneously demonstrate the potential use of flow cytometry for differential identification of molecules from organisms which are in extremely close contact, such as host and parasite. The fact that the parasite ER-like protein differs from its homologue in mammalian cells supports two very important aspects of this study: (a) the parasite ER-like molecule is not a product from host immune or epithelial cells, and (b) although the two proteins differ in some characteristics, they probably conserve a high degree of similarity in their catalytic domains and are thus probably detectable with the same antibody. It is important to mention that the ER-like protein was not only detected at the mRNA and protein levels, but it was also localized inside the parasite cell. Interestingly, parasite cells expressing ER-like protein were exclusively located in the subtegumental tissue, where most of the muscle and nephridium cells are found. This suggests that the ER-like protein is also involved in parasite motility and excretion, as well as in reproductive functions, the two of which together are responsible for maintaining parasite viability and proliferation. Our hypothesis supports the fact that the expression of parasite proteins which can recognize the host’s growth factors represents an advantage in parasite metabolism economy, since the pathogen does not need to synthesize all molecules involved in a pathway, but can use them directly from its host. This benefits the processes of reproduction, establishment and immune evasion, among other important aspects of the parasite’s life.

Regarding the significance of the differential effects of sex hormones on host and parasite, it is interesting to speculate on the evolutionary impact of host gender distinctive specialization in dealing with the parasite’s developmental stages. Thus, it would appear that gender specialization as the one described here for T. crassiceps (females favoring and males hindering asexual larval reproduction) has contributed to the evolution of a more stable host–parasite relationship. Furthermore, this protein in T. crassiceps showed high degree of relation to their ER-␣ counterparts in fish and amphibian, but it is distant to mammalian sequences. This finding has two important connotations: first, it suggests that ER from T. crassiceps is a close relative of the steroid nuclear receptors that bind to estrogens. Second, this ER in T. crassiceps definitively is not a contamination product from mouse or human cells because it has a far relation to ERs sequenced in these organisms. In conclusion, a functional ER-like protein from T. crassiceps is presently described. This ER-like molecule showed great capacity to transduce signals induced by 17-␤-estradiol in the parasite. These results provide further evidence on the mechanism of how the host’ microenvironment affects parasite metabolism. Furthermore, the evolutionary origin of the molecules described herein, which involves the use of the host’s hormones by the parasite, is worth studying. Whether the genes that code for these molecules were acquired by the parasite through horizontal gene transfer or evolved by mimicry, or simply from common ancestral genes, remains to be elucidated. Finally, our findings provide evidence on the cross-talk between host and parasite at molecular and evolutionary levels, and they corroborate the sexual dimorphism of the immune response, providing new information which may be useful in designing anti-helminth drugs able to combat parasite cells specifically with minimal secondary effects to the host. Acknowledgements Financial support was provided by Grant # IN213108 from Programa de Apoyo a Proyectos de Innovación Tecnológica, Dirección General de Asuntos del Personal Académico, (PAPIIT, DGAPA), UNAM to J. Morales-Montor. Romel Hernández-Bello holds a postdoctoral fellowship from DGAPA, UNAM., and Elizabeth G. Ibarra-Coronado holds a scholarship from CONACYT, México. Isabel Pérez Montfort corrected the English version of this manuscript. References [1] Olster DH, Blaustein JD. Development of steroid-induced lordosis in female guinea pigs: effects of different estradiol and progesterone treatments, clonidine, and early weaning. Horm Behav 1989;23:118–29. [2] Pfaff DW, Freidin MM, Wu-Peng XS, Yin J, Zhu YS. Competition for DNA steroid response elements as a possible mechanism for neuroendocrine integration. J Steroid Biochem Mol Biol 1994;49:373–9. [3] Camacho-Arroyo I, Guerra-Araiza C, Dominguez R, Mendoza-Rodriguez CA, Cruz ME, Cerbon MA. C-fos expression pattern in the hypothalamus and the preoptic area of the rat during proestrus. Life Sci 1998;62:1153–9. [4] Guerra-Araiza C, Cerbon MA, Morimoto S, Camacho-Arroyo I. Progesterone receptor isoforms expression pattern in the rat brain during the estrous cycle. Life Sci 2000;66:1743–52. [5] Choi KC, Jeung EB. The biomarker and endocrine disruptors in mammals. J Reprod Dev 2003;49:337–45. [6] An SJ, Zhang YX. Estrogen receptor subtypes and the regulatory effect of receptor ligand binding on gene transcription. Sheng Li Ke Xue Jin Zhan 2002;33:309–12. [7] Okada A, Sato T, Ohta Y, Iguchi T. Sex steroid hormone receptors in the developing female reproductive tract of laboratory rodents. J Toxicol Sci 2005;30:75–89. [8] Charitidi K, Meltser I, Tahera Y, Canlon B. Functional responses of estrogen receptors in the male and female auditory system. Hear Res 2009;252:71–8. [9] Greene GL, Shiau AK, Nettles KW. A structural explanation for ERalpha/ERbeta SERM discrimination. Ernst Schering Res Found Workshop 2004:33–45. [10] Henke BR, Heyer D. Recent advances in estrogen receptor modulators. Curr Opin Drug Discov Dev 2005;8:437–48.

E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149–1159 [11] Morales-Montor J, Chavarria A, De Leon MA, Del Castillo LI, Escobedo EG, Sanchez EN, et al. Host gender in parasitic infections of mammals: an evaluation of the female host supremacy paradigm. J Parasitol 2004;90:531–46. [12] Escobedo G, Larralde C, Chavarria A, Cerbon MA, Morales-Montor J. Molecular mechanisms involved in the differential effects of sex steroids on the reproduction and infectivity of Taenia crassiceps. J Parasitol 2004;90:1235–44. [13] Vargas-Villavicencio JA, Larralde C, Morales-Montor J. Treatment with dehydroepiandrosterone in vivo and in vitro inhibits reproduction, growth and viability of Taenia crassiceps metacestodes. Int J Parasitol 2008;38:775–81. [14] Escobedo G, Camacho-Arroyo I, Hernandez-Hernandez OT, Ostoa-Saloma P, Garcia-Varela M, Morales-Montor J. Progesterone induces scolex evagination of the human parasite Taenia solium: evolutionary implications to the host–parasite relationship. J Biomed Biotechnol 2010;2010:591079. [15] Escobedo G, Soldevila G, Ortega-Pierres G, Chavez-Rios JR, Nava K, FonsecaLinan R, et al. A new MAP kinase protein involved in estradiol-stimulated reproduction of the helminth parasite Taenia crassiceps. J Biomed Biotechnol 2010;2010:747121. [16] Vargas-Villavicencio JA, Larralde C, De Leon-Nava MA, Escobedo G, MoralesMontor J. Tamoxifen treatment induces protection in murine cysticercosis. J Parasitol 2007;93:1512–7. [17] Townson S, Tagboto SK. In vitro cultivation and development of Onchocerca volvulus and Onchocerca lienalis microfilariae. Am J Trop Med Hyg 1996;54:32–7. [18] Morales-Montor J, Escobedo G, Rodriguez-Dorantes M, Tellez-Ascencio N, Cerbon MA, Larralde C. Differential expression of AP-1 transcription factor genes c-fos and c-jun in the helminth parasites Taenia crassiceps and Taenia solium. Parasitology 2004;129:233–43. [19] Hernandez-Bello R, Bermudez-Cruz RM, Fonseca-Linan R, Garcia-Reyna P, Le Guerhier F, Boireau P, et al. Identification, molecular characterisation and differential expression of caveolin-1 in Trichinella spiralis maturing oocytes and embryos. Int J Parasitol 2008;38:191–202. [20] Vargas-Parada L, Laclette JP. Gene structure of Taenia solium paramyosin. Parasitol Res 2003;89:375–8. [21] Yang J, Yang Y, Gu Y, Li Q, Wei J, Wang S, et al. Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis. Biochem Biophys Res Commun 2008;365:528–33. [22] Erttmann KD, Buttner DW. Immunohistological studies on Onchocerca volvulus paramyosin. Parasitol Res 2009;105:1371–4.

1159

[23] Jiz M, Friedman JF, Leenstra T, Jarilla B, Pablo A, Langdon G, et al. Immunoglobulin E (IgE) responses to paramyosin predict resistance to reinfection with Schistosoma japonicum and are attenuated by IgG4. Infect Immun 2009;77:2051–8. [24] Liu H, Miller MS, Swank DM, Kronert WA, Maughan DW, Bernstein SI. Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster. Proc Natl Acad Sci USA 2005;102:10522–7. [25] Larralde C, Morales J, Terrazas I, Govezensky T, Romano MC. Sex hormone changes induced by the parasite lead to feminization of the male host in murine Taenia crassiceps cysticercosis. J Steroid Biochem Mol Biol 1995;52: 575–80. [26] Morales-Montor J, Baig S, Hallal-Calleros C, Damian RT. Taenia crassiceps: androgen reconstitution of the host leads to protection during cysticercosis. Exp Parasitol 2002;100:209–16. [27] Zahner H, Sani BP, Shealy YF, Nitschmann A. Antifilarial activities of synthetic and natural retinoids in vitro. Trop Med Parasitol 1989;40:322–6. [28] Morales-Montor J, Mohamed F, Ghaleb AM, Baig S, Hallal-Calleros C, Damian RT. In vitro effects of hypothalamic–pituitary–adrenal axis (HPA) hormones on Schistosoma mansoni. J Parasitol 2001;87:1132–9. [29] Fantappie MR, Galina A, Luis de Mendonca R, Furtado DR, Secor WE, Colley DG, et al. Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone. Mol Cell Biochem 1999;202:149–58. [30] Damian RT. Parasite immune evasion and exploitation: reflections and projections. Parasitology 1997;115(Suppl):S169–75. [31] Unnasch TR, Bradley J, Beauchamp J, Tuan R, Kennedy MW. Characterization of a putative nuclear receptor from Onchocerca volvulus. Mol Biochem Parasitol 1999;104:259–69. [32] Rajan TV, Ganley L, Paciorkowski N, Spencer L, Klei TR, Shultz LD. Brugian infections in the peritoneal cavities of laboratory mice: kinetics of infection and cellular responses. Exp Parasitol 2002;100:235–47. [33] Maroto M, Arredondo JJ, San Roman M, Marco R, Cervera M. Analysis of the paramyosin/miniparamyosin gene. Miniparamyosin is an independently transcribed, distinct paramyosin isoform, widely distributed in invertebrates. J Biol Chem 1995;270:4375–82.