SAFETY AND PHARMACOKINETICS OF PRAZIQUANTEL IN EUROPEAN POND TURTLES (EMYS ORBICULARIS).

Spirorchiidosis, caused by blood flukes of the genus Spirorchis, is a disease of great concern for the critically endangered European pond turtle (EPT; Emys orbicularis) in Switzerland. The endogenous life cycle of the parasite often leads to systemic inflammatory reactions, thrombosis, and death. Praziquantel (PZQ) is the treatment of choice against adult Spirorchis spp. in green (Chelonia mydas) and in loggerhead (Caretta caretta) sea turtles and is therefore considered for the treatment of EPT. This study aimed to establish a safe, easily applicable PZQ treatment for EPT, based on pharmacokinetics and tolerability. Three application methods were tested in a total of 12 adult EPT. Each turtle received a total of 75 mg/kg PZQ (three doses of 25 mg/kg in 3-h intervals [q3h × 3]) via IM (n = 3 turtles), SC (n = 3 turtles), or PO (n = 6 turtles) administration. Blood was collected 3, 6, 24, and 48 h after the first administration to determine the plasma concentration of PZQ using high-performance liquid chromatography coupled to mass spectrometry. Maximum measured R-PZQ concentrations (Cmax) were reached after 6 h. The mean Cmax of the total PZQ (sum of R- and S-PZQ) in the PO-treated EPT group was 1,929 ng/ml. Significantly higher concentrations were measured after IM and SC injection (mean Cmax of total PZQ = 12,715 ng/ml and 10,114 ng/ml, respectively). Transient side effects were evident after IM administration (local swelling and lameness), whereas no adverse drug effects were observed after PO and SC administration. Based on these results and the ease of administration to EPT, SC injection of PZQ at 25 mg/kg q3h times 3 serves as promising treatment application for the future.

Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

In Vitro Activities of Dithiocarbamate Derivatives against Echinococcus multilocularis Metacestode Vesicles.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the severe zoonotic disease alveolar echinococcosis. New treatment options are urgently needed. Disulfiram and dithiocarbamates were previously shown to exhibit activity against the trematode Schistosoma mansoni. As both parasites belong to the platyhelminths, here we investigated whether these compounds were also active against E. multilocularis metacestode vesicles in vitro. We used an in vitro drug-screening cascade for the identification of novel compounds against E. multilocularis metacestode vesicles with disulfiram and 51 dithiocarbamates. Five compounds showed activity against E. multilocularis metacestode vesicles after five days of drug incubation in a damage marker release assay. Structure-activity relationship analyses revealed that a S-2-hydroxy-5-nitro benzyl moiety was necessary for anti-echinococcal activity, as derivatives without this group had no effect on E. multilocularis metacestode vesicles. The five active compounds were further tested for potential cytotoxicity in mammalian cells. For two compounds with low toxicity (Schl-32.315 and Schl-33.652), IC50 values in metacestode vesicles and IC50 values in germinal layer cells were calculated. The compounds were not highly active on isolated GL cells with IC50 values of 27.0 ± 4.2 µM for Schl-32.315 and 24.7 ± 11.5 µM for Schl-33.652, respectively. Against metacestode vesicles, Schl-32.315 was not very active either with an IC50 value of 41.6 ± 3.2 µM, while Schl-33.652 showed a low IC50 of 4.3 ± 1 µM and should be further investigated in the future for its activity against alveolar echinococcosis.

The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure-Activity Relationships at TRPMPZQ Orthologs.

The anthelmintic drug praziquantel remains a key clinical therapy for treating various diseases caused by parasitic flatworms. The parasite target of praziquantel has remained undefined despite longstanding usage in the clinic, although a candidate ion channel target, named TRPMPZQ, has recently been identified. Intriguingly, certain praziquantel derivatives show different activities against different parasites: for example, some praziquantel analogs are considerably more active against cestodes than against schistosomes. Here we interrogate whether the different activities of praziquantel analogs against different parasites are also reflected by unique structure-activity relationships at the TRPMPZQ channels found in these different organisms. To do this, several praziquantel analogs were synthesized and functionally profiled against schistosome and cestode TRPMPZQ channels. Data demonstrate that structure-activity relationships are closely mirrored between parasites and their TRPMPZQ orthologs, providing further support for TRPMPZQ as the therapeutically relevant target of praziquantel.

Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto.

Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis.

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1'681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Endogenous IL-33 Accelerates Metacestode Growth during Late-Stage Alveolar Echinococcosis.

During the course of the infectious disease alveolar echinococcosis (AE), the larval stage of Echinococcus multilocularis develops in the liver, where an initial Th1/Th17 immune response may allow its elimination in resistant individuals. In patients susceptible to infection and disease, the Th2 response initiates later, inducing tolerance to the parasite. The role of interleukin 33 (IL-33), an alarmin released during necrosis and known to drive a Th2 immune response, has not yet been described during AE. Wild-type (WT) and IL-33-/- C57BL/6J mice were infected by peritoneal inoculation with E. multilocularis metacestodes and euthanized 4 months later, and their immune response were analyzed. Immunofluorescence staining and IL-33 enzyme-linked immunosorbent assay (ELISA) were also performed on liver samples from human patients with AE. Overall, metacestode lesions were smaller in IL-33-/- mice than in WT mice. IL-33 was detected in periparasitic tissues, but not in mouse or human serum. In infected mice, endogenous IL-33 modified peritoneal macrophage polarization and cytokine profiles. Th2 cytokine concentrations were positively correlated with parasite mass in WT mice, but not in IL-33-/- mice. In human AE patients, IL-33 concentrations were higher in parasitic tissues than in distant liver parenchyma. The main sources of IL-33 were CD31+ endothelial cells of the neovasculature, present within lymphoid periparasitic infiltrates together with FOXP3+ Tregs. In the murine model, periparasitic IL-33 correlated with accelerated parasite growth putatively through the polarization of M2-like macrophages and release of immunosuppressive cytokines IL-10 and transforming growth factor ß1 (TGF-ß1). We concluded that IL-33 is a key alarmin in AE that contributes to the tolerogenic effect of systemic Th2 cytokines. IMPORTANCE Infection with the metacestode stage of Echinococcus multilocularis, known as alveolar echinococcosis, is the most severe cestodosis worldwide. However, less than 1% of exposed individuals, in which the immune system is unable to control the parasite, develop the disease. The factors responsible for this interindividual variability are not fully understood. In this in vivo study comparing wild-type and IL-33-/- infected mice, together with data from human clinical samples, we determined that IL-33, an alarmin released following tissue injury and involved in the pathogenesis of cancer and asthma, accelerates the progression of the disease by modulating the periparasitic microenvironment. This suggests that targeting IL-33 could be of interest for the management of patients with AE, and that IL-33 polymorphisms could be responsible for increased susceptibility to AE.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc 1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc 1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity.

The immunomodulatory potential of Trichinella spiralis muscle larvae excretory-secretory products (ES L1) has been well documented in vitro on dendritic cells (DCs) and in animal models of autoimmune diseases. ES L1 products possess the potential to induce tolerogenic DCs and consequently trigger regulatory mechanisms that maintain immune homeostasis. The use of ES L1 as a potential treatment for various inflammatory disorders proved to be beneficial in animal models, although the precise immunomodulatory factors have not yet been identified. This study aimed at the isolation and characterization of ES L1 components that possess galectin family member properties. Galectin-1-like proteins (TsGal-1-like) were isolated from ES L1 based on the assumption of the existence of a lactose-specific carbohydrate-recognition domain and were recognized by anti-galectin-1 antibodies in Western blot. This TsGal-1-like isolate, similar to galectin-1, induced DCs with tolerogenic properties and hence, the capacity to polarize T cell response towards a regulatory type. This was reflected by a significantly increased percentage of CD4+CD25+Foxp3+ regulatory T cells and significantly increased expression of IL-10 and TGF-ß within this cell population. Proteomic analysis of TsGal-1-like isolate by mass spectrometry identified nineteen proteins, seven with annotated function after blast analysis against a database for T. spiralis and the UniProt database. To our surprise, none of the identified proteins possesses homology with known galectin family members. Nevertheless, the isolated components of ES L1 possess certain galectin-1 properties, such as specific lactose binding and the potential to elicit a regulatory immune response, so it would be worth further investigating the structure of sugar binding within isolated proteins and its biological significance.

Albendazole reduces hepatic inflammation and endoplasmic reticulum-stress in a mouse model of chronic Echinococcus multilocularis infection.

BACKGROUND: Echinococcus multilocularis causes alveolar echinococcosis (AE), a rising zoonotic disease in the northern hemisphere. Treatment of this fatal disease is limited to chemotherapy using benzimidazoles and surgical intervention, with frequent disease recurrence in cases without radical surgery. Elucidating the molecular mechanisms underlying E. multilocularis infections and host-parasite interactions ultimately aids developing novel therapeutic options. This study explored an involvement of unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection in mice. METHODS: E. multilocularis- and mock-infected C57BL/6 mice were subdivided into vehicle, albendazole (ABZ) and anti-programmed death ligand 1 (αPD-L1) treated groups. To mimic a chronic infection, treatments of mice started six weeks post i.p. infection and continued for another eight weeks. Liver tissue was then collected to examine inflammatory cytokines and the expression of UPR- and ERS-related genes. RESULTS: E. multilocularis infection led to an upregulation of UPR- and ERS-related proteins in the liver, including ATF6, CHOP, GRP78, ERp72, H6PD and calreticulin, whilst PERK and its target eIF2α were not affected, and IRE1α and ATF4 were downregulated. ABZ treatment in E. multilocularis infected mice reversed, or at least tended to reverse, these protein expression changes to levels seen in mock-infected mice. Furthermore, ABZ treatment reversed the elevated levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the liver of infected mice. Similar to ABZ, αPD-L1 immune-treatment tended to reverse the increased CHOP and decreased ATF4 and IRE1α expression levels. CONCLUSIONS AND SIGNIFICANCE: AE caused chronic inflammation, UPR activation and ERS in mice. The E. multilocularis-induced inflammation and consecutive ERS was ameliorated by ABZ and αPD-L1 treatment, indicating their effectiveness to inhibit parasite proliferation and downregulate its activity status. Neither ABZ nor αPD-L1 themselves affected UPR in control mice. Further research is needed to elucidate the link between inflammation, UPR and ERS, and if these pathways offer potential for improved therapies of patients with AE.

Evaluation of the PrioCHECK™ Trichinella AAD kit to detect Trichinella spiralis, T. britovi, and T. pseudospiralis larvae in pork using the automated digestion method Trichomatic-35.

Trichinellosis is a potentially deadly parasitic zoonosis that is contracted by consuming undercooked infected meat. Reliable detection of infectious Trichinella spp. larvae in meat is therefore pivotal to ensure consumer's safety. The recently authorised PrioCHECK™ Trichinella Alternative Artificial Digestion (AAD) test kit appears promising when used with the standard magnetic stirrer method, but evaluation with other apparatus types is lacking. In this study, the performance of the AAD kit in an adapted Trichomatic-35 (TM35) instrument was evaluated, first, at the Swiss National Reference Laboratory for trichinellosis (NRL); second, in a ring trial involving four Swiss official laboratories. Proficiency pork samples spiked with larvae of Trichinella spiralis, T. britovi, or T. pseudospiralis were tested with the AAD kit and with the reference pepsin-HCl digestion method in TM35 instruments. At the NRL, both methods yielded identical qualitative and similar quantitative results independently of the Trichinella species. In the ring trial, satisfactory results were obtained for 47/50 (94.0%) (AAD) and 62/67 (92.5%) (reference method) of the analysed samples. Technical problems impairing analysis were more frequently observed with the AAD kit (n = 22) than with the reference method (n = 5) and were mainly (16/22) reported by one of the external labs. When no technical issues were recorded, the performance of both methods was comparable, in agreement with the observations at the NRL; however, these results suggest a need for further training with the kit and standardisation of the adapted TM35 instruments.

Maca against Echinococcosis?-A Reverse Approach from Patient to In Vitro Testing.

Drug-based treatment of alveolar echinococcosis (AE) with benzimidazoles is in most cases non-curative, thus has to be taken lifelong. Here, we report on a 56-year-old male AE patient who received standard benzimidazole treatment and biliary plastic stents, and additionally self-medicated himself with the Peruvian plant extract Maca (Lepidium meyenii). After 42 months, viable parasite tissue had disappeared. Based on this striking observation, the anti-echinococcal activity of Maca was investigated in vitro and in mice experimentally infected with Echinococcus multilocularis metacestodes. Albendazole (ABZ)-treated mice and mice treated with an ABZ+Maca combination exhibited a significantly reduced parasite burden compared to untreated or Maca-treated mice. As shown by a newly established UHPLC-MS/MS-based measurement of ABZ-metabolites, the presence of Maca during the treatment did not alter ABZ plasma levels. In vitro assays corroborated these findings, as exposure to Maca had no notable effect on E. multilocularis metacestodes, and in cultures of germinal layer cells, possibly unspecific, cytotoxic effects of Maca were observed. However, in the combined treatments, Maca inhibited the activity of ABZ in vitro. While Maca had no direct anti-parasitic activity, it induced in vitro proliferation of murine spleen cells, suggesting that immunomodulatory properties could have contributed to the curative effect seen in the patient.

Impact on Bile Acid Concentrations by Alveolar Echinococcosis and Treatment with Albendazole in Mice.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis is a chronic, progressive liver disease widely distributed in the Northern Hemisphere. The main treatment options include surgical interventions and chemotherapy with benzimidazole albendazole (ABZ). To improve the current diagnosis and therapy of AE, further investigations into parasite-host interactions are needed. This study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess serum and liver tissue bile acid profiles in the i.p. chronic E. multilocularis-infected mouse model and evaluated the effects of the anthelmintic drug ABZ. Additionally, hepatic mRNA and protein expression of enzymes and transporters regulating bile acid concentrations were analyzed. AE significantly decreased unconjugated bile acids in serum and liver tissue. Taurine-conjugated bile salts were unchanged or increased in the serum and unchanged or decreased in the liver. Ratios of unconjugated to taurine-conjugated metabolites are proposed as useful serum markers of AE. The expression of the bile acid synthesis enzymes cytochrome P450 (CYP) 7A1 and aldo-keto reductase (AKR) 1D1 tended to decrease or were decreased in mice with AE, along with decreased expression of the bile acid transporters Na+/taurocholate cotransporting polypeptide (NTCP) and bile salt efflux pump (BSEP). Importantly, treatment with ABZ partially or completely reversed the effects induced by E. multilocularis infection. ABZ itself had no effect on the bile acid profiles and the expression of relevant enzymes and transporters. Further research is needed to uncover the exact mechanism of the AE-induced changes in bile acid homeostasis and to test whether serum bile acids and ratios thereof can serve as biomarkers of AE and for monitoring therapeutic efficacy.

Innate and adaptive immune responses following PD-L1 blockade in treating chronic murine alveolar echinococcosis.

BACKGROUND: Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) immune checkpoint blockade are efficacious in certain cancer therapies. OBJECTIVES: The present study aimed to provide a picture about the development of innate and adaptive immune responses upon PD-L1 blockade in treating chronic murine AE. METHODS: Immune treatment started at 6 weeks post-E. multilocularis infection, and was maintained for 8 weeks with twice per week anti-PD-L1 administration (intraperitoneal). The study included an outgroup-control with mice perorally medicated with albendazole 5 d/wk, and another one with both treatments combined. Assessment of treatment efficacy was based on determining parasite weight, innate and adaptive immune cell profiles, histopathology and liver tissue cytokine levels. RESULTS/CONCLUSIONS: Findings showed that the parasite load was significantly reduced in response to PD-L1 blockade, and this blockade (a) contributed to T-cell activity by increasing CD4+ /CD8+ effector T cells, and decreasing Tregs; (b) had the capacity to restore DCs and Kupffer cells/Macrophages; (c) suppressed NKT and NK cells; and thus (d) lead to an improved control of E. multilocularis infection in mice. This study suggests that the PD-L1 pathway plays an important role by regulating adaptive and innate immune cells against E. multilocularis infection, with significant modulation of tissue inflammation.

Short communication: Efficacy of albendazole in Echinococcus multilocularis-infected mice depends on the functional immunity of the host.

Alveolar echinococcosis (AE) is a deadly parasitic disease that requires lifelong treatment with albendazole. Development of host immunity is pivotal with regard to the clinical outcome of AE, but its influence on conventional albendazole treatment is unknown. Using T-cell deficient athymic nude mice, we demonstrated that functional immunity is required for albendazole to be efficacious against murine AE. These results call for attention given the increasing number of immunocompromised patients with AE.

Drug repurposing applied: Activity of the anti-malarial mefloquine against Echinococcus multilocularis.

The current chemotherapeutical treatment against alveolar echinococcosis relies exclusively on benzimidazoles, which are not parasiticidal and can induce severe toxicity. There are no alternative treatment options. To identify novel drugs with activity against Echinococcus multilocularis metacestodes, researchers have studied potentially interesting drug targets (e.g. the parasite's energy metabolism), and/or adopted drug repurposing approaches by undertaking whole organism screenings. We here focus on drug screening approaches, which utilize an in vitro screening cascade that includes assessment of the drug-induced physical damage of metacestodes, the impact on metacestode viability and the viability of isolated parasite stem cells, structure-activity relationship (SAR) analysis of compound derivatives, and the mode of action. Finally, once in vitro data are indicative for a therapeutic window, the efficacy of selected compounds is assessed in experimentally infected mice. Using this screening cascade, we found that the anti-malarial mefloquine was active against E. multilocularis metacestodes in vitro and in vivo. To shed more light into the mode of action of mefloquine, SAR analysis on mefloquine analogues was performed. E. multilocularis ferritin was identified as a mefloquine-binding protein, but its precise role as a drug target remains to be elucidated. In mice that were infected either intraperitoneally with metacestodes or orally with eggs, oral treatment with mefloquine led to a significant reduction of parasite growth compared to the standard treatment with albendazole. However, mefloquine was not acting parasiticidally. Assessment of mefloquine plasma concentrations in treated mice showed that levels were reached which are close to serum concentrations that are achieved in humans during long-term malaria prophylaxis. Mefloquine might be applied in human AE patients as a salvage treatment. Future studies should focus on other repurposed anti-infective compounds (MMV665807, niclosamide, atovaquone), which showed stronger in vitro activity against E. multilocularis than mefloquine.

In vitro metabolomic footprint of the Echinococcus multilocularis metacestode.

Alveolar echinococcosis (AE) is a zoonotic disease that is deadly if left untreated. AE is caused by the larval metacestode stage of the cestode Echinococcus multilocularis. Better knowledge on the host-parasite interface could yield novel targets for improvement of the treatment against AE. We analyzed culture media incubated with in vitro grown E. multilocularis metacestodes by 1H nuclear magnetic resonance spectroscopy to identify the unknown metabolic footprint of the parasite. Moreover, we quantitatively analyzed all amino acids, acetate, glucose, lactate, and succinate in time-course experiments using liquid chromatography and enzymatic assays. The E. multilocularis metacestodes consumed glucose and, surprisingly, threonine and produced succinate, acetate, and alanine as major fermentation products. The metabolic composition of vesicle fluid (VF) from in vitro grown E. multilocularis metacestodes was different from parasite-incubated culture medium with respect to the abundance, but not the spectrum, of metabolites, and some metabolites, in particular amino acids, accumulated in the VF. Overall, this study presents the first characterization of the in vitro metabolic footprint of E. multilocularis metacestodes and VF composition, and it provides the basis for analyses of potentially targetable pathways for future drug development.