Alveolar echinococcosis (AE) in humans is a parasitic disease characterized by severe damage to the liver and occasionally other organs. AE is caused by infection with the metacestode (larval) stage of the fox tapeworm Echinococcus multilocularis, usually infecting small rodents as natural intermediate hosts. Conventionally, human AE is chemotherapeutically treated with mebendazole or albendazole. There is, however still the need for improved chemotherapeutical options. Primary in vivo studies on drugs of interest are commonly performed in small laboratory animals such as mice and Mongolian jirds, and in most cases, a secondary infection model is used, whereby E. multilocularis metacestodes are directly injected into the peritoneal cavity or into the liver. Disadvantages of this methodological approach include risk of injury to organs during the inoculation and, most notably, a limitation in the macroscopic (visible) assessment of treatment efficacy. Thus, in order to monitor the efficacy of chemotherapeutical treatment, animals have to be euthanized and the parasite tissue dissected. In the present study, mice were infected with E. multilocularis metacestodes through the subcutaneous route and were then subjected to chemotherapy employing albendazole. Serological responses to infection were comparatively assessed in mice infected by the conventional intraperitoneal route. We demonstrate that the subcutaneous infection model for secondary AE facilitates the assessment of the progress of infection and drug treatment in the live animal.
Alveolar echinococcosis is a disease which affects humans and inflicts severe damage to the liver and other organs. It is caused by a parasite whose definitive host is the fox. Despite being a relatively rare disease, an increasing number of new cases has been reported in central and eastern European countries more recently. The current therapy in human AE patients consists of benzimidazoles. The treatment has to be taken on a daily basis for very long periods of time, or even lifelong. New options are currently being searched for, mainly based on compounds that show efficacy in experimental animal infection models. The infection is commonly done by injecting parasites directly into the peritoneal cavity of the animals, with risk of damage to the surrounding organs. The efficacy of applied treatments can only be evaluated at the end of the studies by dissection of the animals. In this study we show that the subcutaneous infection model can be applied for drug treatment trials and enables the direct monitoring of treatment effects during the entire study period.
The current chemotherapy of alveolar echinococcosis (AE) is based on benzimidazoles such as albendazole and has been shown to be parasitostatic rather than parasiticidal, requiring lifelong duration. Thus, new and more efficient treatment options are urgently needed. By employing a recently validated assay based on the release of functional phosphoglucose isomerase (PGI) from dying parasites, the activities of 26 dicationic compounds and of the (+)- and (−)-erythro-enantiomers of mefloquine were investigated. Initial screening of compounds was performed at 40 μM, and those compounds exhibiting considerable antiparasitic activities were also assessed at lower concentrations. Of the dicationic drugs, DB1127 (a diguanidino compound) with activities comparable to nitazoxanide was further studied. The activity of DB1127 was dose dependent and led to severe structural alterations, as visualized by electron microscopy. The (+)- and (−)-erythro-enantiomers of mefloquine showed similar dose-dependent effects, although higher concentrations of these compounds than of DB1127 were required for metacestode damage. In conclusion, of the drugs investigated here, the diguanidino compound DB1127 represents the most promising compound for further study in appropriate in vivo models for Echinococcus multilocularis infection.
Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis and causes severe disease in the human liver, and occasionally in other organs, that is fatal when treatment is unsuccessful. The present chemotherapy against AE is based on mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some instances, is parasitostatic rather than parasiticidal, and usually involves the lifelong uptake of large doses of drugs. Thus, new treatment options are urgently needed. In this study we investigated the in vitro and in vivo efficacy of mefloquine against E. multilocularis metacestodes. Treatment using mefloquine (20 μM) against in vitro cultures of metacestodes resulted in rapid and complete detachment of large parts of the germinal layer from the inner surface of the laminated layer within a few hours. The in vitro activity of mefloquine was dependent on the dosage. In vitro culture of metacestodes in the presence of 24 μM mefloquine for a period of 10 days was parasiticidal, as determined by murine bioassays, while treatment with 12 μM was not. Oral application of mefloquine (25 mg/kg of body weight administered twice a week for a period of 8 weeks) in E. multilocularis-infected mice was ineffective in achieving any reduction of parasite weight, whereas treatment with albendazole (200 mg/kg/day) was highly effective. However, when the same mefloquine dosage was applied intraperitoneally, the reduction in parasite weight was similar to the reduction seen with oral albendazole application. Combined application of both drugs did not increase the treatment efficacy. In conclusion, mefloquine represents an interesting drug candidate for the treatment of AE, and these results should be followed up in appropriate in vivo studies.
In vitro treatment of Echinococcus multilocularis and Echinococcus granulosus larval stages with the antimalarials dihydroartemisinin and artesunate (10 to 40 μM) exhibited promising results, while 6 weeks of in vivo treatment of mice infected with E. multilocularis metacestodes (200 mg/kg of body weight/day) had no effect. However, combination treatments of both drugs with albendazole led to a substantial but statistically not significant reduction in parasite weight compared to results with albendazole alone.
Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.
The in vitro effects of 19 dicationic diamidine derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750, and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest 50% inhibitory concentrations were found for DB786 (0.21 μM against Neospora and 0.22 μM against Toxoplasma) and DB750 (0.23 μM against Neospora and 0.16 μM against Toxoplasma), with complete proliferation inhibition at 1.7 μM for both drugs against both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected human foreskin fibroblast (HFF) cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. For true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7 μM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFFs to 1.7 μM DB750 for 6, 12, or 24 h, followed by infection with N. caninum tachyzoites and subsequent culture in the absence of DB750, resulted in significantly delayed parasite proliferation. This suggests that either (i) these compounds or their respective active metabolites were still present after the removal of the drugs or (ii) the drug treatments reversibly impaired some functional activities in HFFs that were essential for parasite proliferation and/or survival.
Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis.
The nitrothiazole analogue nitazoxanide [NTZ; 2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] represents the parent compound of a class of drugs referred to as thiazolides and exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. NTZ and other thiazolides are active against a wide range of other intracellular and extracellular protozoan parasites in vitro and in vivo, but their mode of action and respective subcellular target(s) have only recently been investigated. In order to identify potential targets of NTZ and other thiazolides in Giardia lamblia trophozoites, we have developed an affinity chromatography system using the deacetylated derivative of NTZ, tizoxanide (TIZ), as a ligand. Affinity chromatography on TIZ-agarose using cell extracts of G. lamblia trophozoites resulted in the isolation of an approximately 35-kDa polypeptide, which was identified by mass spectrometry as a nitroreductase (NR) homologue (EAA43030.1). NR was overexpressed as a six-histidine-tagged recombinant protein in Escherichia coli, purified, and then characterized using an assay for oxygen-insensitive NRs with dinitrotoluene as a substrate. This demonstrated that the NR was functionally active, and the protein was designated GlNR1. In this assay system, NR activity was severely inhibited by NTZ and other thiazolides, demonstrating that the antigiardial activity of these drugs could be, at least partially, mediated through inhibition of GlNR1.
Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.
The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 μM; in comparison, NTZ and tizoxanide had IC50s of 2.4 μM, and MTZ had an IC50 of 7.8 μM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.
The thiazolide nitazoxanide [2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] (NTZ) exhibits a broad spectrum of activities against a wide variety of intestinal and tissue-dwelling helminths, protozoa, and enteric bacteria infecting animals and humans. The drug has been postulated to act via reduction of its nitro group by nitroreductases, including pyruvate ferredoxin oxidoreductase. In this study, we investigated the efficacies of nitazoxanide and a number of other thiazolides against Neospora caninum tachyzoites in vitro. We employed real-time-PCR-based monitoring of tachyzoite adhesion, invasion, and intracellular proliferation, as well as electron microscopic visualization of the effects imposed by nitazoxanide. In addition, we investigated several modified versions of this drug. These modifications included on one hand the replacement of the nitro group on the thiazole ring with a bromide, thus removing the most reactive group, and on the other hand the differential positioning of methyl groups on the salicylate ring. We show that the thiazole-associated nitro group is not necessarily required for the action of the drug and that methylation of the salicylate ring can result in complete abrogation of the antiparasitic activity, depending on the positioning of the methyl group. These findings indicate that other mechanisms besides the proposed mode of action involving the pyruvate ferredoxin oxidoreductase enzyme could be responsible for the wide spectrum of antiparasitic activity of NTZ and that modifications in the benzene ring could be important in these alternative mechanisms.
The apicomplexan parasite Toxoplasma gondii is a leading opportunistic pathogen associated with AIDS and congenital birth defects. Due to the need for identifying new parasite-specific treatments, the possibility of targeting sphingolipid biosynthesis in the parasite was investigated. Aureobasidin A, an inhibitor of the enzyme synthesizing the sphingolipid inositol phosphorylceramide, which is present in fungi, plants, and some protozoa but absent in mammalian cells, was found to block in vitro T. gondii replication without affecting host cell metabolism. Aureobasidin A treatment did not induce tachyzoite to bradyzoite stage conversion in T. gondii but resulted in a loss of intracellular structures and vacuolization within the parasite. In addition, aureobasidin A inhibited sphingolipid synthesis in T. gondii. Sphingolipid biosynthetic pathways may therefore be considered targets for the development of anti-T. gondii agents.
Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by α-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37°C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced Mr but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.
Echinococcus multilocularis metacestodes are fluid-filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the livers of infected individuals. Tumor-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody (MAb) E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction of Echinococcus granulosus, to characterize potentially related components in E. multilocularis. Immunofluorescence studies demonstrated that MAb E492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. The respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The MAb E492/G1-reactive fraction in E. multilocularis, named Em492 antigen, was isolated by immunoaffinity chromatography. Em492 antigen had a protein/carbohydrate ratio of 0.25, reacted with a series of lectins, and is related to the laminated layer-associated Em2(G11) antigen. The epitope recognized by MAb E492/G1 was sensitive to sodium periodate but was not affected by protease treatment. Anti-Em492 immunoglobulin G1 (IgG1) and IgG2 and, at lower levels, IgG3 were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on concanavalin A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492 antigen, but trypan blue exclusion tests and transmission electron microscopy failed to reveal any cytotoxic effect in Em492 antigen-treated spleen cells. This indicated that Em492 antigen could be modulating the periparasitic cellular environment during E. multilocularis infection through as yet unidentified mechanisms and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface.
We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.
When humans serve as inadvertent intermediate hosts for Echinococcus multilocularis, disease (alveolar echinococcosis [AE]) may result from the expanding parasite metacestode in visceral organs, mostly in the liver. Benzimidazole carbamate derivatives such as mebendazole and albendazole are used for chemotherapeutic treatment of AE. However, these treatments are, in most cases, parasitistatic rather than parasiticidal. As treatment is discontinued, a recurrence of parasite growth has been observed in many AE patients with nonradical resections. The only curative treatment for AE is radical surgical resection of the parasite tissue and support by chemotherapy. As there is a need for new treatment options for AE, the in vitro efficacy of nitazoxanide (NTZ), a broad-spectrum drug used against intestinal parasites and bacteria, was investigated. We showed that in vitro treatment of E. multilocularis metacestodes with NTZ induced high levels of alkaline phosphatase activity in the medium. Concurrently, distinct morphological and ultrastructural alterations were detected. Most significantly, two distinct types of alterations were observed as soon as after 3 h of NTZ treatment. At first, the drug induced a peripheral output of membranous vesicles from the tegumental membrane into the laminated layer. Simultaneously, germinal layer-associated undifferentiated cells produced large vacuoles filled with lipid-like and often electron-dense membranous segments. Other alterations were observed at later time points, including vacuolization of the germinal layer, accumulation of lipid droplets, and lastly, loss of microtriches and separation of the laminated and germinal layers. The pattern of damage induced by NTZ was different from the alterations earlier observed in albendazole sulfoxide-treated vesicles. The nonviability of NTZ-treated metacestodes was confirmed through bioassay, i.e., inoculation of treated and untreated parasites into mice. These experiments demonstrate the in vitro parasiticidal effect of NTZ on E. multilocularis metacestodes.
The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.
In apicomplexan parasites, host cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules. Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the host cell and thus mediate and establish the physical interaction between the parasite and the host cell surface. This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date. NcMIC3 is a microneme-associated protein found in Neospora caninum tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains. We determined the subcellular localization of NcMIC3 prior to and following host cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following host cell lysis in a temperature-dependent manner. Surface-exposed NcMIC3 could be detected up to 2 to 3 h following host cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes. In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system. Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants. Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of N. caninum tachyzoites to adhere to Vero cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains. NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero cells showed that the protein binds to the Vero cell surface. In addition, the EGF-like domains, expressed as recombinant proteins in Escherichia coli, also interacted with the Vero cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient. Our data are indicative of a functional role of NcMIC3 in host cell infection.
Echinococcus multilocularis causes alveolar echinococcosis, one of the most lethal helminthic (accidental) infections in humans, as the life cycle predominantly includes wildlife rodents as intermediate hosts. The physical barrier between the proliferating parasitic metacestode and the host tissue is the acellular laminated layer (LL), which is characterized by its rich high-molecular-weight polysaccharide composition. Conversely to a crude protein-rich vesicular fluid antigen, a major carbohydrate antigen of the LL—the Em2(G11) antigen—did not stimulate murine T-cell proliferation in vitro. In fact, the persistent metacestode growth and antigenic stimulation induced a Th2 shift in vivo following conventional infection by intraperitoneal inoculation of 100 metacestode vesicles into C57/BL6 mice. Concurrently, the expression of Th1 cytokines (interleukin-2 and gamma interferon) remained persistently low until the late stage of chronic infection. In comparison to a recombinant proteinic II/3 antigen, the specific immunoglobulin G (IgG) response against the Em2(G11) antigen (including all IgG isotypes) maintained persistently low avidity. Furthermore, the Em2(G11) antigen induced a specific IgM and IgG response in T-cell-deficient athymic nude, TCRβ−/−, major histocompatibility complex class II (MHCII)−/−(CD4-deficient), and CD40−/− mice. The Em2(G11)-specific IgG synthesized in nude TCRβ−/− and MHCII−/− mice was predominantly of the IgG3 and IgG2a isotypes and of the IgG3 and IgG2b isotypes in CD40−/− mice. This finding suggested that in vivo, the IgG response to major carbohydrate antigen Em2(G11) of E. multilocularis could take place independently of αβ+ CD4+ T cells and in the absence of CD40-CD40 ligand interactions; thus, the Em2(G11) antigen of the acellular LL represents a T-cell-independent antigen. Functionally, the encapsulating LL, and especially its major carbohydrate antigen, Em2(G11), seems to be one of the key factors in the parasite's survival strategy and acts by modulating the host immune response by virtue of its T-cell-independent nature.
Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. The disease affects the human liver and occasionally other organs and is fatal if treatment is unsuccessful. The present chemotherapy of AE is based on the administration of benzimidazole carbamate derivatives, such as mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some cases, parasitostatic rather than parasiticidal, and the recurrence rate is rather high. Therefore, chemotherapy usually involves the lifelong uptake of massive doses of albendazole and new treatment options are urgently needed. In order to avoid costly and time-consuming animal experimentation, a first step in searching for novel parasiticidal compounds could be the in vitro drug screening of novel compounds by employing metacestode cultivation. However, presently used techniques (e.g., transmission electron microscopy) for determination of parasite viability involve costly equipment and time-consuming preparation of rather large amounts of parasite material. We therefore searched for a parasite marker which can be easily traced and the presence or absence of which is indicative of parasite viability. In this study we show that the increase of E. multilocularis alkaline phosphatase activity in culture supernatants during in vitro drug treatment with albendazole derivatives correlates with the progressive degeneration and destruction of the metacestode tissue. The inexpensive and rapid assay presented here will serve as an ideal tool for performing first-round in vitro tests on the efficacy of a large number of antiparasitic compounds.
The metacestode stage of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a parasitic disease affecting the liver, with occasional metastasis into other organs. Benzimidazole carbamate derivatives such as mebendazole and albendazole are currently used for chemotherapeutic treatment of AE. Albendazole is poorly resorbed and is metabolically converted to its main metabolite albendazole sulfoxide, which is believed to be the active component, and further to albendazole sulfone. Chemotherapy with albendazole has been shown to have a parasitostatic rather than a parasitocidal effect; it is not effective in all cases, and the recurrence rate is rather high once chemotherapy is stopped. Thus, development of new means of chemotherapy of AE is needed. This could include modifications of benzimidazoles and elucidiation of the respective biological pathways. In this study we performed in vitro drug treatment of E. multilocularis metacestodes with albendazole sulfoxide and albendazole sulfone. High-performance liquid chromatography analysis of vesicle fluids showed that the drugs were taken up rapidly by the parasite. Transmission electron microscopic investigation of parasite tissues and nuclear magnetic resonance spectroscopy of vesicle fluids demonstrated that albendazole sulfoxide and albendazole sulfone had similar effects with respect to parasite ultrastructure and changes in metabolites in vesicle fluids. This study shows that the in vitro cultivation model presented here provides an ideal first-round test system for screening of antiparasite drugs.
The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of ∼65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.
African trypanosomes are not passively transmitted, but they undergo several rounds of differentiation and proliferation within their intermediate host, the tsetse fly. At each stage, the survival and successful replication of the parasites improve their chances of continuing the life cycle, but little is known about specific molecules that contribute to these processes. Procyclins are the major surface glycoproteins of the insect forms of Trypanosoma brucei. Six genes encode proteins with extensive glutamic acid–proline dipeptide repeats (EP in the single-letter amino acid code), and two genes encode proteins with an internal pentapeptide repeat (GPEET). To study the function of procyclins, we have generated mutants that have no EP genes and only one copy of GPEET. This last gene could not be replaced by EP procyclins, and could only be deleted once a second GPEET copy was introduced into another locus. The EP knockouts are morphologically indistinguishable from the parental strain, but their ability to establish a heavy infection in the insect midgut is severely compromised; this phenotype can be reversed by the reintroduction of a single, highly expressed EP gene. These results suggest that the two types of procyclin have different roles, and that the EP form, while not required in culture, is important for survival in the fly.