Progesterone in vitro increases growth, motility and progesterone receptor expression in third stage larvae of Toxocara canis.

The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.

Proteomic profile associated with cell death induced by androgens in Taenia crassiceps cysticerci: proposed interactome.

Androgens have been shown to exert a cysticidal effect upon Taenia crassiceps, an experimental model of cysticercosis. To further inquire into this matter, the Taenia crassiceps model was used to evaluate the expression of several proteins after testosterone (T4) and dihydrotestosterone (DHT) in vitro treatment. Under 2-D proteomic maps, parasite extracts were resolved into approximately 130 proteins distributed in a molecular weight range of 10-250 kDa and isoelectrical point range of 3-10. The resultant proteomic pattern was analysed, and significant changes were observed in response to T4 and DHT. Based on our experience with electrophoretic patterns and proteomic maps of cytoskeletal proteins, alteration in the expression of isoforms of actin, tubulin and paramyosin and of other proteins was assessed. Considering that androgens may exert their biological activity in taeniids through the non-specific progesterone receptor membrane component (PGRMC), we harnessed bioinformatics to propose the identity of androgen-regulated proteins and establish their hypothetical physiological role in the parasites. These analyses yield a possible explanation of how androgens exert their cysticidal effects through changes in the expression of proteins involved in cytoskeletal rearrangement, dynamic vesicular traffic and transduction of intracellular signals.

Dihydrotestosterone enhances growth and infectivity of Leishmania Mexicana.

A strong sex-associated susceptibility towards Leishmania has been reported in males, yet little is known on the effect of hormones in Leishmania physiopathogenicity. Due to the enhanced susceptibility of males to Leishmania mexicana infections, we were interested in analysing the effect exerted by the main androgen produced in males (DHT) on L. mexicana promastigotes. Thus, the aim of this study was to assess the regulation exerted by dihydrotestosterone (DHT) on L. mexicana replication, infectivity, survival and development of tissue lesions. Experiments included growth curves of L. mexicana promastigotes incubated with different doses of DHT, their infection rate, intracellular survival and lesion development in BALB/c mice. Our data show that DHT significantly enhances parasite replication, infection rate and survival in bone marrow-derived macrophages (BMMФ). Promastigotes in the presence of DHT produced significantly larger lesions in BALB/c earlobes. These results suggest that DHT probably plays a critical role during L. mexicana infections, and the higher susceptibility of males possibly relates to benefits gained by the parasite from host-derived hormones. Our data shed new light on the physiopathology of Leishmania infections and are the first attempt to understand the direct interaction between Leishmania and androgens, particularly DHT. Understanding this trans-regulation process employed by parasites to exploit host molecules sheds new light on L. mexicana physiopathogenesis and opens a possible field for studies on drug development.

Toxicokinetic assessment of inhalatory absorption of Diisobutyl phthalate (DiBP) using a novel thermal desorption-GC-MS method to determine phthalate diesters in blood plasma.

Phthalates are endocrine disrupting compounds that have been found in outdoor and indoor air. However, little is known about their inhalatory absorption. Although measurement of urinary metabolites is the current standard, complex and convergent metabolism of phthalates poses the necessity for alternative methodologies such as the quantitation of parental compounds in plasma. We determined the inhalatory absorption of Diisobutyl phthalate (DiBP) using a novel method based on a thermal desorption probe (TSP)-gas chromatography-mass spectrometry developed for the detection and quantitation of nine phthalate diesters in blood plasma, which fulfilled the acceptance criteria suggested by FDA guidelines regarding specificity, matrix effect, recovery, linearity, sensitivity, accuracy, and precision. After inhalation, plasma concentration of DiBP exhibited two peaks, suggesting a first, rapid absorption event, followed by a second, delayed one and a first order elimination stage. Half-life was calculated as 62 min and bioavailability, compared to IV route, was 15%.

Immunodiagnosis of neurocysticercosis: ways to focus on the challenge.

Neurocysticercosis (NCC) is a disease of the central nervous system that is considered a public health problem in endemic areas. The definitive diagnosis of this disease is made using a combination of tools that include imaging of the brain and immunodiagnostic tests, but the facilities for performing them are usually not available in endemic areas. The immunodiagnosis of NCC is a useful tool that can provide important information on whether a patient is infected or not, but it presents many drawbacks as not all infected patients can be detected. These tests rely on purified or semipurified antigens that are sometimes difficult to prepare. Recent efforts have focused on the production of recombinant or synthetic antigens for the immunodiagnosis of NCC and interesting studies propose the use of new elements as nanobodies for diagnostic purposes. However, an immunodiagnostic test that can be considered as "gold standard" has not been developed so far. The complex nature of cysticercotic disease and the simplicity of common immunological assumptions involved explain the low scores and reproducibility of immunotests in the diagnosis of NCC. Here, the most important efforts for developing an immunodiagnostic test of NCC are listed and discussed. A more punctilious strategy based on the design of panels of confirmed positive and negative samples, the use of blind tests, and a worldwide effort is proposed in order to develop an immunodiagnostic test that can provide comparable results. The identification of a set of specific and representative antigens of T. solium and a thorough compilation of the many forms of antibody response of humans to the many forms of T. solium disease are also stressed as necessary.

Altered expression of cytokines and sex steroid receptors in the reproductive tract of cysticercotic male mice.

Infection with Taenia crassiceps cysticerci in male mice produces an increase in serum oestradiol levels, whereas serum testosterone is abolished. Concomitantly, complete atrophy of the reproductive tract of infected male mice is observed. The present study was undertaken to determine the expression pattern of cytokines involved in steroidogenesis and sex steroid receptors in the reproductive tissues of normal and infected male mice, and relating this expression pattern to whole parasite counts, serum sex steroid levels and pathology of the reproductive tract in infected male mice. The expression of IL-4, IFN-gamma and TNF-alpha in testes and seminal vesicles was markedly increased in infected mice; however, IL-10 and IL-1beta expression was importantly decreased in the same organs. IL-2 expression in reproductive tissues was not affected by infection. The infection markedly induced the expression of androgen receptor, in both reproductive organs tested, while subtypes of oestrogen receptors were decreased in both tissues.

Immunoendocrine host-parasite interactions during helminth infections: from the basic knowledge to its possible therapeutic applications.

Most of the current research on parasitic infections that affect humans and domestic animals has been focused on vaccines, diagnostic methods, epidemiology, new drug design, and recently, with the advancement of genomics and proteomics, on the evolutionary origins of parasites. However, the basic biology of many parasites of medical and veterinary importance has not been intensively studied. Some efforts have been made to obtain information on the parasite-host relationship; however, knowledge of the intricate neuroimmunoendocrine interactions of the host-parasite network, the consequences of this interaction on the host and parasite physiology, and its possible applications needs further investigation. We review here the literature, our own studies on the host-parasite neuroimmunoendocrine network, and how this basic knowledge can be used to design new treatments, by way of using hormones, antihormones, and hormone analogues as a possible novel therapy during parasitic diseases, with special emphasis on helminth parasites. Besides the biological interest, these investigations may contribute to the future identification of alternative treatments for several parasitic diseases. This complicated neuroimmunoendocrine network management during parasitic infections, and its physiological and behavioural consequences upon the host, may be operative in other mammalian infections. Such complexity may also help to explain the often conflicting results, observed between infections with respect to the role of the host sex and age, and hints to other avenues of research and strategies for their treatment and control.

Gonadectomy inhibits development of experimental amoebic liver abscess in hamsters through downregulation of the inflammatory immune response.

Incidence of amoebic liver abscess (ALA) in human males is considerably higher than in females, suggesting a role for sex hormones in this parasite infection. We describe here the effect of hamster gonadectomization on the development of ALA. After monitoring the decrease of oestradiol in females and testosterone in males to undetectable levels by ELISA and Radio Immuno Assay (RIA) in serum, hamsters were intraportally infected with Entamoeba histolytica trophozoites and killed 7 days later. ALA was absent in 50% of male and 15% of female gonadectomized (Gdx) hamsters, in comparison with 100% infection in non-Gdx controls. This protection against ALA in Gdx hamsters was concomitant to a comparatively scarce inflammatory infiltrate and necrosis surrounding clusters of trophozoites in the liver tissue, as well as to a lack of response of spleen cells to Con A, evaluated in proliferation assays. As tissue damage in ALA has been associated with a local inflammatory Th1 response, we determined the profile of response in hamsters by immunohistochemistry on liver sections. In contrast to strong Th1 responses in non-Gdx animals, Gdx females and males exhibited Th2 and Th3 profiles of cytokines, respectively, suggesting that protection against ALA following gonadectomization, could be related to downregulation of liver Th1 response during amoebic infection.

The role of cytokines in the regulation of neurotransmission.

Cytokines are highly inducible, secretory proteins that mediate intercellular communication in the immune system. They are grouped in several protein families, namely tumor necrosis factors, interleukins, interferons and colony-stimulating factors. In recent years, evidence has elucidated that some of these proteins as well as their receptors are also produced in the central nervous system (CNS) by specific neural cell lineages under physiological and pathological conditions. Cytokines regulate a variety of processes in the CNS, including neurotransmission. The current data let us to suggest that cytokines play an important role in the regulation of both excitatory and inhibitory neurotransmission in the CNS. This knowledge could be fundamental for the proposal of new therapeutic approaches to neurological and psychiatric disorders.

Differential in vitro effects of insulin on Taenia crassiceps and Taenia solium cysticerci.

Hormones play a significant role in murine cysticercosis (Taenia crassiceps), and increase the frequency of porcine cysticercosis caused by Taenia solium. In the present study, we report the in vitro effect of insulin on the larval stages of T. crassiceps (ORF strain) and T. solium. In vitro exposure of T. crassiceps cysticerci to insulin was found to stimulate this parasite's reproduction twofold with respect to control values, while the same treatment had no effect on T. solium cysticerci. Moreover, normal female mice (BALB/cAnN) infected with T. crassiceps cysticerci previously exposed to insulin presented larger parasite loads than mice inoculated with vehicle-treated cysticerci. To determine the possible molecular mechanisms by which insulin affects T. crassiceps, the insulin receptor was amplified by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Interestingly, both T. crassiceps and T. solium expressed the insulin receptor, although insulin had effects only on T. crassiceps. These results demonstrate that insulin has a dichotomistic effect; it acts directly only on T. crassiceps cysticerci reproduction, possibly through its binding to a specific insulin receptor synthesized by the parasite. Thus, insulin may be recognized by T. crassiceps cysticercus cells as a mitogenic factor, and contribute to parasite proliferation inside the host, as well as to the female mouse susceptibility to T.crassiceps. This phenomenon has not been reported for cysticercosis caused by T. solium, which could, in part, be related to the poor effect of insulin upon the human parasite.

Impact of naturally acquired Taenia solium cysticercosis on the hormonal levels of free ranging boars.

In chronically infected BALBc/AnN male mice, Taenia crassiceps cysticercosis induces changes in the host's sex steroids hormone that lead to their estrogenization and deandrogenization, with possible repercussions on their susceptibility to infections. Here reported are the serum steroid levels in free range cysticercotic male boars. Therefore, the possible effects of Taenia solium cysticerci over the pig steroid levels were evaluated. Herein are described the sex steroids and cortisol levels of non-cysticercotic (n=25) and cysticercotic (n=22) adult boars, as diagnosed by tongue inspection, all free-ranging in a typical village of an endemic rural area in Mexico. A significant reduction of testosterone (P=0.022) and a likely one of 17beta-estradiol (P=0.08) levels were found in the cysticercotic boars in comparison with those non-cysticercotic, whilst no significant differences in the cortisol and DHEA levels were detected. Serum levels of specific antibodies did not correlate with infection nor with the levels of any of the hormones measured. Results suggest that T. solium cysticercosis significantly affects the hormonal status of its porcine host independently of their antibody response.

Progesterone inhibits the in vitro L3/L4 molting process in Haemonchus contortus.

We evaluated the direct effects of progesterone on the morphology, maturation and behavior of Haemonchus contortus larvae in vitro. The presence and location of possible progesterone receptors in these larvae were also determined. The addition of 8ng/mL of progesterone to larval cultures over 10days reduced larval enlargement, while the addition of 160ng/mL of the hormone increased the enlargement. Up to 62% and 65% of the H. contortus larvae molted from third-stage larvae (L3) to fourth-stage larvae (L4) when cultured in RPMI-1640 media without hormone for 5 and 10days, respectively. The addition of different progesterone concentrations (1, 8, 16, 80 and 160ng/mL) to the larval cultures significantly inhibited the molting process within the same periods. The addition of 8ng/mL or higher progesterone concentrations to the cultures significantly increased larval motility (p<0.05) compared with unstimulated larvae. Flow cytometry showed the expression of progesterone receptors (P4-R) in 15% of the cells from newly isolated H. contortus larvae. When the larvae were cultured for 5days in the presence of the hormone, the percentage of P4-R+ cells remained the same. In contrast, unstimulated larvae showed a significant reduction in the number of P4-R+ cells. Using confocal microscopy, a greater concentration of P4-Rs was immunolocated in the anterior portion of the alimentary tract of the larvae, suggesting that the cells in this region are targeted by the hormone. The results of the present study show that H. contortus larvae have possible P4-Rs and respond to this hormone by inhibiting their molting process, thereby suggesting the participation of progesterone in the larval arrest phenomenon.

Use of near infrared fluorescence during robot-assisted laparoscopic partial nephrectomy.

BACKGROUND: Partial nephrectomy is the treatment of choice for T1a tumours. The open approach is still the standard method. Robot-assisted laparoscopic surgery offers advantages that are applicable to partial nephrectomy, such as the use of the Firefly® system with near-infrared fluorescence. OBJECTIVE: To demonstrate the implementation of fluorescence in nephron-sparing surgery. CASE REPORT: This case concerned a 37-year-old female smoker, with obesity. The patient had a right kidney tumour measuring 31 mm, which was found using tomography. She therefore underwent robot-assisted laparoscopic partial nephrectomy, with a warm ischaemia time of 22 minutes and the use of fluorescence with the Firefly® system to guide the resection. There were no complications. The tumour was a pT1aN0M0 renal cell carcinoma, with negative margins. Robot-assisted renal laparoscopic surgery is employed for nephron-sparing surgery, with good oncological and functional results. The combination of the Firefly® technology and intraoperative ultrasound can more accurately delimit the extent of the lesion, increase the negative margins and decrease the ischaemia time. CONCLUSION: Near-infrared fluorescence in robot-assisted partial nephrectomy is useful for guiding the tumour resection and can potentially improve the oncological and functional results.

The in vitro effect of prolactin on the growth, motility and expression of prolactin receptors in larvae of Toxocara canis.

The in vitro effect of prolactin (PRL) on the growth and motility of Toxocara canis larvae was assessed. Additionally, the expression and location of prolactin receptors (PRL-Rs) were determined in the larvae. Larvae of T. canis were incubated with different concentrations of PRL for different periods of time. The stimulated larvae accelerated their enlargement and increased their motility. The mean percentage of PRL-R+ cells in non-stimulated larvae, measured by flow cytometry was 7.3±0.3%. Compared with non-stimulated larvae, the mean fluorescence intensity (p<0.05) increased in larvae incubated with 40ng/mL of PRL for 10 days. A 465-bp length fragment was amplified from larvae gDNA by PCR. The sequence of this fragment showed 99% similarity with the gene fragment that codes for the PRL-R of the domestic dog. A high concentration of PRL-Rs was immune-located in the posterior region of the larval intestine; therefore, the intestinal cells in this region were most likely the targets for this hormone. Based on these results, PRL-Rs were identified in T. canis larvae, and the in vitro stimulation with PRL increased the number of these receptors, accelerated the growth and modified the activity of larvae. All of the above suggest that T. canis larvae are evolutionarily adapted to recognize the PRL of their definitive host and furthermore might explain the reactivation of tissue-arrested larvae during the gestation of bitches, which does not occur in gestating females of other species.

Inhibition of p-450 aromatase prevents feminisation and induces protection during cysticercosis.

Cysticercotic male mice undergo an impressive feminisation process, characterised by 200 times increased serum 17beta-estradiol levels while testosterone and dihydrotestosterone are 90% reduced, which results in elevated parasite burden. Administration of Fadrozole (an aromatase inhibitor) in male and female mice suppressed the production of 17beta-estradiol, accompanied with a 70% reduction in parasite burden. This protective effect was associated in male mice with a recovery of the specific cellular immune response. Interleukin-6 (IL-6) serum levels, and its production by splenocytes, was augmented by 80%, together with a 10-fold increase in its expression in testes of infected male mice. Fadrozole treatment returned these levels to baseline values. Aromatase expression in the testes of infected male mice was not affected by Fadrozole. These results suggest that aromatase and IL-6 are key molecules in the production of the feminisation undergone by infected male mice and to Fadrozole treatment as a possible new therapeutic approach to cysticercosis.

Tissue damage in the male murine reproductive system during experimental Taenia crassiceps cysticercosis.

Chronic infection with Taenia crassiceps cysticerci in male mice increases the level of estradiol in serum, whereas it reduces that of testosterone. In addition, male mice lose their typical male reproductive behavior. The effects of cysticerci infection on the histomorphology of male reproductive tissues are unknown. The present study was undertaken to determine the histological changes in testes, seminal vesicles, and prostate of male mice infected with T. crassiceps cysticerci. At 16 wk of infection, all tissues exhibited high inflammatory infiltrate. Tissue lesions included marked dilation and peripheral fibrosis. In the testes, a diminution of spermiogenesis was observed. The overall results indicated that the histological changes in chronically parasitized male mice occurred with changes in hormone levels, simultaneously with the high inflammatory immune response.

In vitro effects of hypothalamic-pituitary-adrenal axis (HPA) hormones on Schistosoma mansoni.

The effects of in vitro treatment of cercariae, schistosomula, and adult worms of Schistosoma mansoni with 4 hypothalamic-pituitary-adrenal (HPA) axis hormones are described. Dehydroepiandrosterone (DHEA) had the strongest effect on viability. Cercariae were more susceptible to this hormone than schistosomula and adults. Mechanically transformed schistosomula showed 100% mortality (determined microscopically by progressive internal disorganization, development of lucent areas in the cytoplasm, and progressive loss of motility) after 48 hr, whereas physiologically induced schistosomula were more resistant, maintaining viability for up to 5 days of exposure. Males were considerably less sensitive than females to the lethal action of DHEA. When adult worms were paired, DHEA lethality was markedly reduced, with viability beginning to decrease only after 4 days in culture. Cortisol reduced the viability of each of the stages tested about equally. Corticotropin-releasing hormone (CRH) and adrenocorticotropin (ACTH) did not affect the viability of any stage. DHEA and cortisol significantly inhibited in vitro oviposition, whereas CRH and ACTH did not. DHEA and cortisol exerted their effects on schistosome viability and oviposition in a concentration-dependent manner. These results suggest possible new avenues for the control of schistosomiasis.

Host gender in parasitic infections of mammals: an evaluation of the female host supremacy paradigm.

A review of current literature on mammalian hosts' sexual dimorphism (SD) in parasitic infections revealed that (1) it is a scarcely and superficially studied biological phenomenon of considerable significance for individual health, behavior, and lifestyles and for the evolution of species; (2) there are many notable exceptions to the rule of a favorable female bias in susceptibility to infection; (3) a complex network of molecular and cellular reactions connecting the host's immuno-neuroendocrine systems with those of the parasite is responsible for the host-parasite relationship rather than just an adaptive immune response and sex hormones; (4) a lack of gender-specific immune profiles in response to different infections; (5) the direct effects of the host hormones on parasite physiology may significantly contribute to SD in parasitism; and (6) the need to enrich the reductionist approach to complex biological issues, like SD, with more penetrating approaches to the study of cause-effect relationships, i.e., network theory. The review concludes by advising against generalization regarding SD and parasitism and by pointing to some of the most promising lines of research.

Beyond the reproductive effect of sex steroids: their role during immunity to helminth parasite infections.

During the helminth infections, the immune system tends to be modulated by host's sex hormones. Actually, many studies show the reciprocal relationship between sex steroids, the immune system and the elimination or establishment of helminth parasites. Is well known that innate immune response determines the type of adaptive immune response, so the effects in the innate immune response by hormones may affect subsequent adaptive immunity. The sex steroids as estrogens, progesterone and testosterone regulate growth, differentiation, survival and function of many cell types that could be involved in process like homeostasis and immunity, but also have a direct effect on the helminthes, that may probably be mediated by specific receptors on these parasites. Sex steroids, parasites and immunity are closely connected, and their interconnection is involved in the maintenance of elimination or establishment of helminthes in an immunocompetent host. For that reason, understanding the action's mechanisms of sex steroids on immune cells and its direct effect on helminth parasites is important for further progress in the development of novel therapies for chronic helminth diseases associated to immune dysregulation. In this review, we will describe the effects of sex steroids on the immune response during helminth infections as well as the direct effect in these parasites, and the possible implications of these effects on the incidence of several helminth infections.

The neuroimmunoendocrine network in the complex host-parasite relationship during murine cysticercosis.

We review here the role that sex steroids play in experimental intraperitoneal Taenia crassiceps cysticercosis of male and female BalbC/AnN mice. Briefly, estrogens favour and androgens hinder the reproduction of cysticerci by at least two main mechanisms: 1) through estradiol tilting the TH2/TH1 immune system balance towards parasite-permissive TH2 response,which is IL-6 dependent mediating P450-aromatase over expression, shunting testosterone towards estradiol and thus creating a positive feed-back loop which progressively favours TH2 response, blocking in turns TH1 and furthers parasite growth; and 2) estrogens and androgens acting directly upon the cysticercus reproductive system, favoring or hindering, respectively, its asexual reproduction. Later infection, when parasite loads are for milliars, male mice become estrogenized, deandrogenized and diminish their copulative, aggressive and social behaviors in association with P450-aromatase testis overexpression. Changes in c-fos expression in different areas of the infected mice brain point to the additional connection of the central nervous system with the infection driven events, which senses and perhaps reacts to infection with behavioral changes. This complex immune-neuro-endocrine network management of parasite loads in murine cysticercosis, and its physiological and behavioral consequences upon the host, may be operative in other infections of mammals. Such complexity may also help to explain the often conflicting results, observed between infections with respect to the role of the host sex, and hints to other avenues of research and strategies for their treatment and control.