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The larvae-to-adult development on the life cycle of zoonotic parasitic tapeworm Taenia solium involves striking -but clinically unappreciated- events with pivotal importance in cestode biology. Unlike the ones related to the intermediate host, the early-adult stages can be addressed in vitro offering a useful model to study evagination, strobilation and worm development. In the absence of a stage-specific transcriptome, postgenomic data exploration followed by single-gene relative expression analysis by RT-qPCR (reverse transcription-quantitative PCR) are useful strategies to gather information on the regulation of genes of interest during parasite development. However, this approach requires the validation of an endogenous reference gene (RG) to achieve accurate comparisons. Therefore, we analyzed the expression stability of 17 candidate RGs on the context of the early-adult stages of T. solium classified as non-evaginated and evaginated larvae (cysts). The comprehensive tool RefFinder ranked malate dehydrogenase as the most stable gene within these conditions, and its suitability for relative quantification was validated by normalizing the expression of the transporter TGTP1 gene, known for being upregulated upon evagination. This study is the first attempt in finding reliable normalization standards for transcript exploration in genus Taenia.

Excessive erythrocytosis (EE) is the main sign of chronic mountain sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high-altitude populations worldwide. The pathophysiological mechanism of EE is still controversial, as physiological variability of systemic respiratory, cardiovascular, and hormonal responses to chronic hypoxemia complicates the identification of underlying causes. Induced pluripotent stem cells derived from CMS highlanders showed increased expression of genes relevant to the regulation of erythropoiesis, angiogenesis, cardiovascular, and steroid-hormone function that appear to explain the exaggerated erythropoietic response. However, the cellular response to hypoxia in native CMS cells is yet unknown. This study had three related aims: to determine the hypoxic proliferation of native erythroid progenitor burst-forming unit-erythroid (BFU-E) cells derived from CMS and non-CMS peripheral blood mononuclear cells; to examine their sentrin-specific protease 1 (SENP1), GATA-binding factor 1 (GATA1), erythropoietin (EPO), and EPO receptor (EPOR) expression; and to investigate the functional upstream role of SENP1 in native progenitor differentiation into erythroid precursors. Native CMS BFU-E colonies showed increased proliferation under hypoxic conditions compared with non-CMS cells, together with an upregulated expression of SENP1, GATA1, EPOR; and no difference in EPO expression. Knock-down of the SENP1 gene abolished the augmented proliferative response. Thus, we demonstrate that native CMS progenitor cells produce a larger proportion of erythroid precursors under hypoxia and that SENP1 is essential for proliferation. Our findings suggest a significant intrinsic component for developing EE in CMS highlanders at the cellular and gene expression level that could be further enhanced by systemic factors such as alterations in respiratory control, or differential hormonal patterns.

MicroRNAs (miRNAs) are short, endogenous, non-coding, single-stranded RNAs involved in post-transcriptional gene regulation. Although, several miRNAs have been identified in parasitic helminths, there is little information about their identification and function in Taenia. Furthermore, the impact of miRNAs in neurocysticercosis, the brain infection caused by larvae of Taenia solium is still unknown. During chronic infection, T. solium may activate numerous mechanisms aimed to modulate host immune responses. Helminthic miRNAs might also have effects on host mRNA expression and thus play an important role regulating host-parasite interactions. Also, the diagnosis of this disease is difficult and it usually requires neuroimaging and confirmatory serology. Since miRNAs are stable when released, they can be detected in body fluids and therefore have potential to diagnose infection, determine parasite burden, and ascertain effectiveness of treatment or disease progression, for instance. This review discusses the potential roles of miRNAs in T. solium infection, including regulation of host-parasite relationships and their eventual use as diagnostic or disease biomarkers. Additionally, we summarize the bioinformatics resources available for identification of T. solium miRNAs and prediction of their targets.

Background The onset of anthelmintic treatment of neurocysticercosis (NCC) provokes an acute immune response of the host, which in human cases is associated with exacerbation of neurological symptoms. This inflammation can occur at the first days of therapy. So, changes in the brain cysts appearance may be detected by medical imaging. We evaluated radiological changes in the appearance of brain cysts (enhancement and size) on days two and five after the onset of antiparasitic treatment using naturally infected pigs as a model for human NCC. Methods and results Contrast T1-weighted magnetic resonance imaging with gadolinium was performed before and after antiparasitic treatment. Eight NCC-infected pigs were treated with praziquantel plus albendazole and euthanized two (n = 4) and five (n = 4) days after treatment; another group of four infected pigs served as untreated controls. For each lesion, gadolinium enhancement intensity (GEI) and cyst volume were measured at baseline and after antiparasitic treatment. Volume and GEI quantification ratios (post/pre-treatment measures) were used to appraise the effect of treatment. Cysts from untreated pigs showed little variations between their basal and post treatment measures. At days 2 and 5 there were significant increases in GEI ratio compared with the untreated group (1.32 and 1.47 vs 1.01, p = 0.021 and p = 0.021). Cyst volume ratios were significantly lower at days 2 and 5 compared with the untreated group (0.60 and 0.22 vs 0.95, p = 0.04 and p = 0.02). Cysts with lower cyst volume ratios showed more marked post-treatment inflammation, loss of vesicular fluid and cyst wall wrinkling. Conclusion/Significance A significant and drastic reduction of cyst size and increased pericystic enhancement occur in the initial days after antiparasitic treatment as an effect of acute perilesional immune response. These significant changes showed that early anthelmintic efficacy (day two) can be detected using magnetic resonance imaging., Author summary Neurocysticercosis (NCC) is a frequent parasitic infection of the human brain and the most common cause of adult onset epilepsy in developing countries. Acute inflammatory response in NCC plays an important role in the pathogenesis of symptoms by anthelminitic therapies. The anthelmintic recommended therapy for NCC has drawbacks as the exacerbation of inflammation around degenerating cysts provokes the appearance of symptoms at the first days of treatment. Radiological changes in the appearance of cysts usually are seen after months of therapy. To evaluate if significant radiological changes (enhancement and size) occur in the first days of therapy, we used a porcine NCC model and magnetic resonance imaging (MRI) with contrast solution. The major radiological changes observed after treatment with albendazole and praziquantel were an increase in enhancement and the significant reduction in cyst size by day 2 and more evident on day 5. Cysts with greater changes also experienced exacerbated inflammation, loss of vesicular fluid and wrinkling of the cyst wall. These results show an early therapeutic effect and the possible utility of repeat MRI imaging within a few days after starting treatment. Finally, these findings contribute to our understanding of the treatment induced early exacerbation of symptoms.

Neurocysticercosis is a widely prevalent disease in the tropics that causes seizures and a variety into of neurological symptoms in most of the world. Experimental models are limited and do not allow assessment of the degree of inflammation around brain cysts. The vital dye Evans Blue (EB) was injected to 11 pigs naturally infected with Taenia solium cysts to visually identify the extent of disruption of the blood-brain barrier. A total of 369 cysts were recovered from the 11 brains and classified according to the staining of their capsules as blue or unstained. The proportion of cysts with blue capsules was significantly higher in brains from pigs that had received anthelmintic treatment 48 and 120 h before the EB infusion, indicating a greater compromise of the blood-brain barrier due to treatment. The model could be useful for understanding the pathology of treatment-induced inflammation in neurocysticercosis.

Background Disease manifestations in neurocysticercosis (NCC) are frequently due to inflammation of degenerating Taenia solium brain cysts. Exacerbated inflammation post anthelmintic treatment is associated with leakage of the blood brain barrier (BBB) using Evans blue (EB) staining. How well EB extravasation into the brain correlates with magnetic resonance imaging (MRI) using gadolinium (Gd) enhancement as a contrast agent and pericystic inflammation was analyzed in pigs harboring brain cysts of Taenia solium. Methodology/Principal Findings Three groups of 4 naturally infected pigs were assessed. The first and second groups were treated with both praziquantel plus albendazole and sacrificed two and five days post treatment, respectively. A third untreated group remained untreated. Pigs were injected with EB two hours prior to evaluation by Gd-enhanced T1-MRI, and euthanized. The EB staining for each cyst capsule was scored (EB grades were 0: 0%; 1: up to 50%; 2: over 50% but less than 100%; 3: 100%). Similarly, the Gd enhancement around each cyst was qualitatively and quantitatively scored from the MRI. The extent of pericystic inflammation on histology was scored in increasing severity as IS1, IS2, IS3 and IS4. Grade 3 EB staining and enhancement was only seen in treated capsules. Also, treated groups had higher Gd intensity than the untreated group. Grades of enhancement correlated significantly with Gd enhancement intensity. EB staining was correlated with Gd enhancement intensity and with IS4 in the treated groups. These correlations were stronger in internally located cysts compared to superficial cysts in treated groups. Significance EB staining and Gd enhancement strongly correlate. The intensity of enhancement determined by MRI is a good indication of the degree of inflammation. Similarly, EB staining highly correlates with the degree of inflammation and may be applied to study inflammation in the pig model of NCC., Author Summary Neurocysticercosis (NCC) is a frequent parasitic infection of the human brain in developing countries. The symptomatology of human NCC after antiparasitic treatment is generally related to inflammation. The presence and degree of enhancement after intravascular injection of the contrast agent gadolinium in magnetic resonance imaging (MRI) is commonly considered an evidence of blood brain barrier (BBB) leakage. Experimentally, the presence and degree of extravasation of Evans blue (EB) after intravascular injection into the tissues of the brain is a direct measure of blood brain barrier leakage. The BBB leakage of gadolinium in neurocysticercosis is commonly used as an indirect measure of inflammation but has never been experimentally proven. Here we evaluated the relationship between contrast T1-MRI, EB staining and histology findings in naturally infected pigs. There was a strong correlation between EB staining, contrast MRI and histopathology findings after antiparasitic treatment. This correlation was stronger when cysts were internally located in the brain than in superficial cysts partly located in the subarachnoid space (meninges). Contrast-enhanced MRI is a non invasive tool used in diagnosis and follow up of NCC patients. This study shows that the use of EB staining allows for the same conclusions as when using MRI post-treatment, and that both techniques correlate with histopathology findings. These results support the use of EB staining to study NCC using the porcine model as well as validate MRI enhancement to assess brain inflammation in patients.

The 57-residue small hydrophilic endoplasmic reticulum-associated protein (SHERP) shows highly specific, stage-regulated expression in the non-replicative vector-transmitted stages of the kinetoplastid parasite, Leishmania major, the causative agent of human cutaneous leishmaniasis. Previous studies have demonstrated that SHERP localizes as a peripheral membrane protein on the cytosolic face of the endoplasmic reticulum and on outer mitochondrial membranes, whereas its high copy number suggests a critical function in vivo. However, the absence of defined domains or identifiable orthologues, together with lack of a clear phenotype in transgenic parasites lacking SHERP, has limited functional understanding of this protein. Here, we use a combination of biophysical and biochemical methods to demonstrate that SHERP can be induced to adopt a globular fold in the presence of anionic lipids or SDS. Cross-linking and binding studies suggest that SHERP has the potential to form a complex with the vacuolar type H(+)-ATPase. Taken together, these results suggest that SHERP may function in modulating cellular processes related to membrane organization and/or acidification during vector transmission of infective Leishmania.

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections.

Background Neurocysticercosis (NCC), infection of the central nervous system by Taenia solium cysticerci, is a pleomorphic disease. Inflammation around cysticerci is the major cause of disease but is variably present. One factor modulating the inflammatory responses may be the location and characteristics of the brain tissue adjacent to cysticerci. We analyzed and compared the inflammatory responses to cysticerci located in the parenchyma to those in the meninges or cysticerci partially in contact with both the parenchyma and the meninges (corticomeningeal). Methodology/Principal Findings Histological specimens of brain cysticerci (n = 196) from 11 pigs naturally infected with Taenia solium cysticerci were used. Four pigs were sacrificed after 2 days and four after 5 days of a single dose of praziquantel; 3 pigs did not receive treatment. All pigs were intravenously injected with Evans Blue to assess disruption of the blood-brain barrier. The degree of inflammation was estimated by use of a histological score (ISC) based on the extent of the inflammation in the pericystic areas as assessed in an image composed of several photomicrographs taken at 40X amplification. Parenchymal cysticerci provoked a significantly greater level of pericystic inflammation (higher ISC) after antiparasitic treatment compared to meningeal and corticomeningeal cysticerci. ISC of meningeal cysticerci was not significantly affected by treatment. In corticomeningeal cysticerci, the increase in ISC score was correlated to the extent of the cysticercus adjacent to the brain parenchyma. Disruption of the blood-brain barrier was associated with treatment only in parenchymal tissue. Significance Inflammatory response to cysticerci located in the meninges was significantly decreased compared to parenchymal cysticerci. The suboptimal inflammatory response to cysticidal drugs may be the reason subarachnoid NCC is generally refractory to treatment compared to parenchymal NCC., Author Summary The cystic larvae of the pork tapeworm Taenia solium may affect the human brain causing neurocysticercosis (NCC), a very frequent cause of neurological symptoms in developing countries. The clinical expression and response to treatment of human NCC are related to the location of cysticerci inside (intraparenchymal) or outside the brain parenchyma (extraparenchymal NCC). We used a naturally infected pig model to assess the characteristics of inflammation around brain cysticerci of parenchymal, meningeal and mixed locations. There were no major differences in inflammation without treatment. After antiparasitic treatment with praziquantel, inflammation around parenchymal brain cysticerci increased in comparison to meningeal located cysticerci. Cysticerci partially surrounded by both brain parenchyma and meninges showed increased inflammation in relation to the extent of the cysticercus in the brain parenchyma. The location of cysticerci within the brain is a factor that determines the extent and degree of the immune response following anticysticidal treatment. Similar changes may occur in treated human infections. Our work could contribute to explain the differences in response to antiparasitic treatment in different forms of human neurocysticercosis.

Neoblasts are totipotent cells, solely responsible for the proliferation and maturation of tissues in free-living flatworms. Similar cells have been isolated from parasitic flatworms such as Echinococcus. Taenia solium causes human taeniasis (intestinal) and cysticercosis in humans and pigs. Brain infection with larvae (cysts) of T. solium results in neurocysticercosis which is hyperendemic in Peru, and its treatment is associated with serious neurological symptoms. The proliferative capacity and development stages of T. solium have not been described and the neoblasts of this parasite have not been characterized We looked for cell proliferation in T. solium cysts collected from an infected pig, which were identified when replicating and incorporating bromodeoxyuridine nucleotide detected with a monoclonal antibody. A stable cell line of neoblasts would be useful for systematic in vitro studies on drug efficacy and the biology of T. solium. Los neoblastos son células totipotentes, únicas responsables de la proliferación y maduración de tejidos en platelmintos de vida libre. Células similares se han aislado en platelmintos parásitos como Echinococcus. Taenia solium causa la teniasis humana (intestinal) y la cisticercosis en humanos y cerdos. La infección del cerebro con larvas (quistes) de T. solium resulta en neurocisticercosis, hiperendémica en el Perú; su tratamiento se asocia a síntomas neurológicos graves. La capacidad proliferativa y el desarrollo de los estadios de T. solium aún no se describen, y no se ha caracterizado los neoblastos de este parásito. Se buscó células proliferativas en quistes de T. solium colectados de un cerdo infectado, que fueron identificadas al replicarse e incorporar el nucleótido bromodesoxiuridina, detectado con un anticuerpo monoclonal. Una línea celular estable de neoblastos sería útil para estudios sistemáticos in vitro sobre eficacia de drogas y sobre la biología de T. solium.

Cysticidal treatment of neurocysticercosis, an infection of humans and pig brains with Taenia solium, results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations. Treatment-induced pericystic inflammation and its association with blood brain barrier (BBB) dysfunction, as determined by Evans blue (EB) extravasation, was studied in infected untreated and anthelmintic-treated pigs. We compared the magnitude and extent of the pericystic inflammation, presence of EB-stained capsules, the level of damage to the parasite, expression of genes for proinflammatory and regulatory cytokines, chemokines, and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained (blue) and non stained (clear) cysts. Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment. The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours. Expression levels of the proinflammatory genes for IL-6, IFN-γ, TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues, and were generally highest at 120 hr. Additionally, expression of some markers of immunoregulatory activity (IL-10, IL-2Rα) were decreased in EB-stained capsules. An increase in other markers for regulatory T cells (CTLA4, FoxP3) was found, as well as significant increases in expression of two metalloproteases, MMP1 and MMP2 at 48 hr and 120 hr post-treatment. We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response, largely limited to pericystic tissues with compromised vascular integrity. Because treatment induced inflammation occurs in porcine NCC similar to that in human cases, this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation., Author Summary Neurocysticercosis is caused by infection of the brain with the larval (cyst) stage of the tape worm Taenia solium in humans and pigs. Antiparasitic drug treatment is compromised by worsening of neurological symptoms during therapy due to reactive inflammation triggered by the dying parasite. The immune mechanisms that cause this inflammation are poorly understood. In this study, we investigated the nature of inflammation after treatment in pigs naturally infected with T. solium cysts. Evans blue dye injected into infected pigs marks areas in the brain where the normally impermeable capillaries have become more permeable, allowing damaging cells and molecules to leak out into the brain. By microscopy and measurement of gene expression for inflammation-inducing immune mediators, we show that inflammation in the brain tissues around cysts is more severe with increased vessel leakage. Furthermore, the levels of these mediators increased after antiparasitic drug treatment. A significant implication of these findings is that it may be possible to inhibit the inflammation around parasites using drugs or biologics that inhibit these inflammatory pathways and, thereby, reduce local brain damage during treatment. These observations may also be applicable to other inflammatory conditions that affect the brain.

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-( R )- and (−)-( S ) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-( R )- and (−)-( S ) enantiomers against T. solium cysts were evaluated in vitro . Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-( R )-ABZSO was significantly more active than (−)-( S )-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-( R ) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole.

Female sex workers (FSWs) are at high risk of human papillomavirus (HPV) infection. Questionnaires were administered to 200 FSWs aged 18–26 years in Lima, Peru, to gather risk behaviours, and cervical swab samples were collected for Pap smears and HPV DNA testing as part of a longitudinal study. Participants reported a median of 120 clients in the past month, and 99.2% reported using condoms with clients. The prevalence of any HPV in cervical samples was 66.8%; 34 (17.1%) participants had prevalent HPV 16 or 18, and 92 (46.2%) had one or more oncogenic types. Fifteen women had abnormal Pap smears, 13 of which were HPV DNA positive. Fewer years since first sex was associated with oncogenic HPV prevalence in a model adjusted for previous sexually transmitted infection (STI) status and condom use with partners (prevalence ratio = 0.77, 95% confidence interval [CI] = 0.60–0.97). Our data confirm the high rates of HPV transmission among FSWs in Peru, highlighting the need for early and effective strategies to prevent cervical cancer.

The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.

Neurocysticercosis resulting from Taenia solium infections is a major cause of adult-acquired seizures worldwide. Disease is caused by larval cysts, and treatment consists of the anthelmintic drugs albendazole or praziquantel. There are no standard methods to assess drug activity to T. solium cysts in vitro . Morphological, functional, and biochemical changes that might reflect damaging (inhibiting, cytotoxic) drug effects were analyzed after exposure of cysts to albendazole sulfoxide (ABZ-SO), the major active metabolite of the drug in vivo , praziquantel (PZQ), or combinations of both. PZQ exposure led to a decrease in cyst size and inhibition of evagination, whereas ABZ-SO exposure resulted in minimal changes. Alkaline phosphatase (AP) is normally secreted by cysts, and both drugs inhibited AP secretion at concentrations of 5 and 50 ng/ml for PZQ and ABZ-SO, respectively. Some combinations of both drugs resulted in additive and/or synergistic activities. Parasite-specific antigen, detected in the cerebrospinal fluid and blood of infected patients, is also normally secreted by T. solium cysts. Antigen secretion was similarly inhibited by ABZ-SO and PZQ and a combination of both drugs, suggesting that inhibition of secretion is a common downstream consequence of the activities of both drugs. These studies establish quantitative methods to measure in vitro anthelmintic activity and suggest combination therapy with ABZ-SO and PZQ may have clinical benefit.

Transcriptional regulation in Treponema pallidum subsp. pallidum is poorly understood, primarily because this organism cannot be cultivated in vitro or genetically manipulated. We have recently shown a phase variation mechanism controlling transcription initiation of Subfamily II tpr (T. pallidum repeat) genes (tprE, tprG, and tprJ), a group of virulence factor candidates. Furthermore, the same study suggested that additional mechanisms might influence the level of transcription of these tprs. The T. pallidum genome sequence has revealed a few open reading frames (ORFs) with similarity to known bacterial transcription factors (TFs), including four catabolite activator protein (CAP) homologs. In this work, sequences matching the E. coli cAMP receptor protein (CRP) binding motif were identified in silico upstream of tprE, tprG, and tprJ. Using elecrophoretic mobility shift assay (EMSA) and DNaseI footprinting assay, recombinant TP0262, a T. pallidum CRP homolog, was shown to bind specifically to amplicons obtained from the tpr promoters containing putative CRP binding motifs. Using a heterologous reporter system, binding of TP0262 to these promoters was shown to either increase (tprE and tprJ) or decrease (tprG) tpr promoter activity. This is the first characterization of a T. pallidum transcriptional modulator which influences tpr promoter activity.

All kinetoplastids contain membrane-bound microbodies known as glycosomes,in which several metabolic pathways including part of glycolysis are compartmentalized. Peroxin 2 is essential for the import of many proteins into the microbodies of yeasts and mammals. The PEX2 gene of Trypanosoma brucei was identified and its expression was silenced by means of tetracycline-inducible RNA interference. Bloodstream-form trypanosomes, which rely exclusively on glycolysis for ATP generation, died rapidly upon PEX2 depletion. Insect-form (procyclic) trypanosomes do not rely solely on glycolysis for ATP synthesis. PEX2 depletion in procyclic forms resulted in relocation of most tested matrix proteins to the cytosol, and these mutants also died. Compartmentation of microbody enzymes is therefore essential for survival of bloodstream and procyclic T. brucei. In contrast, yeasts and cultured mammalian cells grow normally in the absence of peroxisomal membranes unless placed on selective media.

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian ‘bloodstream form’ trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.