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Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting over 86 million people worldwide. To control and more rapidly eliminate onchocerciasis requires treatments that target the adult stage of the parasite. Drug discovery efforts are challenged by the lack of preclinical animal models using the human-pathogenic filariae, requiring the use of surrogate parasites for Onchocerca volvulus for both ex vivo and in vivo evaluation. Herein, we describe a platform utilizing phenotypic ex vivo assays consisting of the free-living nematode Caenorhabditis elegans, microfilariae and adult filariae of the bovine filariae Onchocerca lienalis and Onchocerca gutturosa, respectively, as well as microfilariae and adult filariae of the feline filariae Brugia pahangi, the rodent filariae Litomosoides sigmodontis and the human-pathogenic filariae Brugia malayi to assess activity across various surrogate parasites. Utilization of those surrogate nematodes for phenotypic ex vivo assays in order to assess activity across various parasites led to the successful establishment of a screening cascade and identification of multiple compounds with potential macrofilaricidal activity and desirable physicochemical, MW = 200-400 and low lipophilicity, logP4, and pharmacokinetic properties, rat and human liver S9 stability of ≥70% remaining at 60 min, and AUC exposures above 3 μM h. This platform demonstrated the successful establishment of a screening cascade which resulted in the discovery of potential novel macrofilaricidal compounds for futher drug discovery lead optimization efforts. This screening cascade identified two distinct chemical series wherein one compound produced a significant 68% reduction of adult Litomosoides sigmodontis in the mouse model. Successful demonstration of efficacy prompted lead optimization medicinal chemistry efforts for this novel series.

Reliable and field suitable serodiagnosis of visceral leishmaniasis (VL) in East Africa has until now been a big challenge due to low sensitivity and cross-reactivity with other pathogens. To improve VL serodiagnosis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed and tested with a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other infectious diseases.

The filarial nematode Onchocerca volvulus causes onchocerciasis (river blindness), a neglected tropical disease affecting 21 million people, mostly in Sub-Saharan Africa. Targeting the endosymbiont Wolbachia with antibiotics leads to permanent sterilization and killing of adult worms. The gold standard to assess Wolbachia depletion is the histological examination of adult worms in nodules beginning at 6 months post-treatment. However, nodules can only be used once, limiting the time points to monitor Wolbachia depletion. A diagnostic to longitudinally monitor Wolbachia depletion from microfilariae (MF) at more frequent intervals wOvftsZ to quantify Wolbachia from as few as one MF that had migrated from skin biopsies and compared quantification using circular and linearized plasmids or synthetic dsDNA (gBlock®). qPCR for MF from the rodent nematode Litomosoides sigmodontis was used to support the reproducibility and validate the principle. The qPCR using as few as 2 MF from O. volvulus and L. sigmodontis reproducibly quantified Wolbachia. Use of a linearized plasmid standard or synthesized dsDNA resulted in numbers of Wolbachia/MF congruent with biologically plausible estimates in O. volvulus and L. sigmodontis MF. The qPCR assay yielded a median of 48.8 (range 1.5–280.5) Wolbachia/O. volvulus MF. The qPCR is a sensitive tool for quantifying Wolbachia in a few MF from skin biopsies and allows for establishing the qPCR as a surrogate parameter for monitoring Wolbachia depletion in adult worms of new antiwolbachial candidates.

Covering: August 1984 up to January 2022Worldwide, increasing morbidity and mortality due to antibiotic-resistant microbial infections has been observed. Therefore, better prevention and control of infectious diseases, as well as appropriate use of approved antibacterial drugs are crucial. There is also an urgent need for the continuous development and supply of novel antibiotics. Thus, identifying new antibiotics and their further development is once again a priority of natural product research. The antibiotic corallopyronin A was discovered in the 1980s in the culture broth of the Myxobacterium

Conventional diagnosis of filarial infections is based on morphological identification of microfilariae using light microscopy and requires considerable expertise, is time-consuming, and can be subjective. Loop-mediated isothermal amplification (LAMP) has advantages over microscopy or PCR because of its operational simplicity, rapidity and versatility of readout options. LAMP assays represent a major step forward in improved filarial diagnostic tools suitable for low resource settings and field applicability. The study goal was to retrospectively evaluate the performance and suitability of the O-150, RF4, and Mp419 LAMP assays for diagnosing Onchocerca volvulus, Loa loa and Mansonella perstans infections, respectively, in humans and vectors under experimental and natural field conditions. Surveys were conducted in four health districts of Cameroon using skin snip and thick blood film methods to detect skin (O. volvulus) and blood (L. loa and M. perstans) dwelling microfilaria in humans. Engorged vectors (Simulium spp., Chrysops spp., and Culicoides spp.) were evaluated by LAMP. Dissected, wild-caught vectors were also analyzed. LAMP showed a prevalence of 40.4% (O. volvulus), 17.8% (L. loa) and 36.6% (M. perstans) versus 20.6% (O. volvulus), 17.4% (L. loa) and 33.8% (M. perstans) with microscopy. Simulium spp. were dissected for microscopy and pooled for LAMP. The O-150 LAMP assay infection rate was 4.3% versus 4.1% by microscopy. Chrysops spp. were dissected and analyzed individually in the LAMP assay. The RF4 LAMP assay infection rate was 23.5% versus 3.3% with microscopy. The RF4 LAMP assay also detected parasites in Chrysops spp. fed on low microfilaremic volunteers. The Mp419 LAMP assay infection rate was 0.2% for C. milnei and 0.04% for C. grahamii, while three other species were LAMP-negative. The sensitivity, species specificity, rapidity and ease of its use of these filarial LAMP assays, and validation of their performance in the field support use as alternatives to microscopy as diagnostic and surveillance tools in global health programs aimed to eliminate onchocerciasis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health Organization’s high priority pathogen organism, with an estimated > 100,000 deaths worldwide in 2019. Thus, there is an unmet medical need for novel and resistance-breaking anti-infectives. The natural product Co-rallopyronin A (CorA), currently in preclinical development for filariasis, is efficacious against MRSA in vitro. In this study, we evaluated the pharmacokinetics of CorA after dosing in mice. Furthermore, we determined compound concentrations in target compartments, such as lung, kidney and thigh tissue, using LC-MS/MS. Based on the pharmacokinetic results, we evaluated the pharmacodynamic profile of CorA using the standard neutropenic thigh and lung infection models. We demonstrate that CorA is effective in both standard pharmacodynamic models. In addition to reaching effective levels in the lung and muscle, CorA was detected at high levels in the thigh bone. The data presented herein encourage the further exploration of the additional CorA indications treatment of MRSA- and methicillin-sensitive S. aureus- (MSSA) related infections.

Oxfendazole is one of the lead macrofilaricidal candidates for the treatment of onchocerciasis and lymphatic filariasis. Originally, oxfendazole was developed for the veterinary market, where it is mainly used to treat intestinal helminth infections. In humans, oxfendazole was proven to be safe in multiple ascending dose studies. Furthermore, previous experimental studies demonstrated that the benzimidazoles class is active in animals and humans against filarial nematodes. In the present study, we have compared the efficacy of oxfendazole isomers with the commercially available racemic mixture Dolthene against the rodent filaria Litomosoides sigmodontis in female BALB/c mice. Treatment with either the isomers or Dolthene led to a reduction of the adult worm burden by 94-98% following the ten-day treatment and by 72% (oxfendazole (-)), 85% (oxfendazole (+)) and 91% (Dolthene) following the five-day treatment. No statistically significant differences in the macrofilaricidal efficacy against L. sigmodontis were observed for both isomers and Dolthene. Metabolites of oxfendazole are fenbendazole and fenbendazole sulfone. Two hours after treatment with Dolthene and both oxfendazole isomers, fenbendazole sulfone, but rarely fenbendazole, was detected. The oxfendazole (-) isomer was metabolised at the highest rate to fenbendazole sulfone. Furthermore, oxfendazole isomers have a comparable pharmacokinetic profile in dogs. In conclusion, our data does not point at the development of a single isomer for future use in humans.

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis., Graphical abstract Image 1, Highlights • Emodepside causes concentration dependent paralysis on filariae. • Findings confirm the broad-spectrum nematicidal profile of emodepside. • Data reveal emodepside as a promising drug candidate for human filariasis.

Two new ircinianin-type sesterterpenoids, ircinianin lactone B and ircinianin lactone C (7 and 8), together with five known entities from the ircinianin compound family (1, 3–6) were isolated from the marine sponge Ircinia wistarii. Ircinianin lactones B and C (7 and 8) represent new ircinianin terpenoids with a modified oxidation pattern. Despite their labile nature, the structures could be established using a combination of spectroscopic data, including HRESIMS and 1D/2D NMR techniques, as well as computational chemistry and quantum-mechanical calculations. In a broad screening approach for biological activity, the class-defining compound ircinianin (1) showed moderate antiprotozoal activity against Plasmodium falciparum (IC50 25.4 μM) and Leishmania donovani (IC50 16.6 μM).

In vivo studies in mice provide a valuable model to test novel active pharmaceutical ingredients due to their low material need and the fact that mice are frequently used as a species for early efficacy models. However, preclinical in vitro evaluations of formulation principles in mice are still lacking. The development of novel in vitro and in silico models supported the preclinical formulation evaluation for the anti-infective corallopyronin A (CorA). To this end, CorA and solubility-enhanced amorphous solid dispersion formulations, comprising povidone or copovidone, were evaluated regarding biorelevant solubilities and dissolution in mouse-specific media. As an acidic compound, CorA and CorA-ASD formulations showed decreased solubilities in mice when compared with human-specific media. In biorelevant biphasic dissolution experiments CorA-povidone showed a three-fold higher fraction partitioned into the organic phase of the biphasic dissolution, when compared with CorA-copovidone. Bioavailabilities determined by pharmacokinetic studies in BALB/c mice correlated with the biphasic dissolution prediction and resulted in a Level C in vitro–in vivo correlation. In vitro cell experiments excluded intestinal efflux by P-glycoprotein or breast cancer resistance protein. By incorporating in vitro results into a physiologically based pharmacokinetic model, the plasma concentrations of CorA-ASD formulations were predicted and identified dissolution as the limiting factor for bioavailability.

Infections with helminths affect more than one billion people worldwide. Despite an urgent need there is no vaccine available that would confer long lasting protection against helminth infections. Previous studies indicated that a vaccination with irradiated infective L3 reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as adjuvant improves the efficacy of a vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in increased neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels at the site of injection. To investigate the in vivo impact on parasite clearance, BALB/c mice received 3 subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Serum analysis before the challenge infection confirmed the induction of L. sigmodontis-specific antibodies in response to the immunization and serum from immunized mice significantly reduced larval motility in vitro with naïve cells. 63 days after the challenge infection, vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a significantly greater reduction in adult worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). Further, the treatment of L. sigmodontis infection with 3pRNA alone, but not poly(I:C), resulted in a reduced worm burden, supporting the therapeutic potential for the activation of RIG-I with 3pRNA. In conclusion, our data demonstrate that the additional activation of nucleic acid sensing immune receptors boosts the immune response and provides better protection against L. sigmodontis. Thus, the use of nucleic acid receptor agonists as vaccine adjuvants represents a promising novel strategy to improve the efficacy of vaccines against filariae and potentially of other helminths.Author SummaryFilarial nematodes can cause debilitating diseases such as onchocerciasis and lymphatic filariasis that present a major public health burden in the tropics and subtropics, putting more than a billion people at risk of infection. Filarial diseases are transmitted to humans by insect vectors as they take a blood meal. The WHO classifies both filarial infections as neglected tropical diseases and aims to eliminate the transmission of onchocerciasis and eliminate lymphatic filariasis as public health problem by 2030. However, up to date there is no vaccination available that could support the efforts to eliminate filarial diseases and potentially helminth infections in general. Here, we used the well-established murine model for filarial infection, Litomosoides sigmodontis, to test the use of nucleic acid receptor agonists as vaccine adjuvants to enhance local immune responses. We found that infection with L. sigmodontis induces type I IFN and our vaccine strategy enhances the production of type I IFN resulting in increased parasite-specific immune responses and enhanced worm clearance. In summary, our study provides a promising novel approach for a vaccination strategy using cytosolic RNA receptor agonists.

Filariae are parasitic roundworms, which can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Lymphatic filariasis, also known as elephantiasis, and onchocerciasis, commonly referred to as river blindness, can lead to stigmatizing pathologies and present a socio-economic burden for affected people and their endemic countries. Filariae typically induce a type 2 immune response, which is characterized by cytokines, i.e., IL-4, IL-5 and IL-13 as well as type 2 immune cells including alternatively activated macrophages, innate lymphoid cells and Th2 cells. However, the hallmark characteristic of filarial infections is a profound eosinophilia. Eosinophils are innate immune cells and pivotal in controlling helminth infections in general and filarial infections in particular. By modulating the function of other leukocytes, eosinophils support and drive type 2 immune responses. Moreover, as primary effector cells, eosinophils can directly attack filariae through the release of granules containing toxic cationic proteins with or without extracellular DNA traps. At the same time, eosinophils can be a driving force for filarial pathology as observed during tropical pulmonary eosinophilia in lymphatic filariasis, in dermatitis in onchocerciasis patients as well as adverse events after treatment of onchocerciasis patients with diethylcarbamazine. This review summarizes the latest findings of the importance of eosinophil effector functions including the role of eosinophil-derived proteins in controlling filarial infections and their impact on filarial pathology analyzing both human and experimental animal studies.

Two new ircinianin-type sesterterpenoids, ircinianin lactone B and ircinianin lactone C (

Group 2 innate lymphoid cells (ILC2s) are inducers of type 2 immune responses, but their role during filarial infection remains unclear. In the present study, we used the Litomosoides sigmodontis rodent model of filariasis to analyze ILC2s during infection in susceptible BALB/c mice that develop a chronic infection with microfilaremia and semi-susceptible C57BL/6 mice that eliminate the filariae shortly after the molt into adult worms and thus do not develop microfilaremia. ILC2s (CD45+ Lineage- TCRβ- CD90.2+ Sca-1+ IL-33R+ GATA-3+) were analyzed in the pleural cavity, the site of L. sigmodontis infection, after the infective L3 larvae reached the pleural cavity (9 days post infection, dpi), after the molt into adult worms (30dpi) and during the peak of microfilaremia (70dpi). C57BL/6 mice had significantly increased ILC2 numbers compared to BALB/c mice at 30dpi, accompanied by substantially higher IL-5 and IL-13 levels, indicating a stronger type 2 immune response in C57BL/6 mice upon L. sigmodontis infection. At this time point the ILC2 numbers positively correlated with the worm burden in both mouse strains. ILC2s and GATA-3+ CD4+ T cells were the dominant source of IL-5 in L. sigmodontis-infected C57BL/6 mice with ILC2s showing a significantly higher IL-5 expression than CD4+ T cells. To investigate the importance of ILC2s during L. sigmodontis infection, ILC2s were depleted with anti-CD90.2 antibodies in T and B cell-deficient Rag2-/- C57BL/6 mice on 26-28dpi and the outcome of infection was compared to isotype controls. Rag2-/- mice were per se susceptible to L. sigmodontis infection with significantly higher worm burden than C57BL/6 mice and developed microfilaremia. Depletion of ILC2s did not result in an increased worm burden in Rag2-/- mice, but led to significantly higher microfilariae numbers compared to isotype controls. In conclusion, our data demonstrate that ILC2s are essentially involved in the control of microfilaremia in Rag2-/- C57BL/6 mice.

Filariae are vector-borne parasitic nematodes that are endemic worldwide, in tropical and subtropical regions. Important human filariae spp. include Onchocerca volvulus, Wuchereria bancrofti and Brugia spp., and Loa loa and Mansonella spp. causing onchocerciasis (river blindness), lymphatic filariasis (lymphedema and hydrocele), loiasis (eye worm), and mansonelliasis, respectively. It is estimated that over 1 billion individuals live in endemic regions where filarial diseases are a public health concern contributing to significant disability adjusted life years (DALYs). Thus, efforts to control and eliminate filarial diseases were already launched by the WHO in the 1970s, especially against lymphatic filariasis and onchocerciasis, and are mainly based on mass drug administration (MDA) of microfilaricidal drugs (ivermectin, diethylcarbamazine, albendazole) to filarial endemic areas accompanied with vector control strategies with the goal to reduce the transmission. With the United Nations Sustainable Development Goals (SDGs), it was decided to eliminate transmission of onchocerciasis and stop lymphatic filariasis as a public health problem by 2030. It was also requested that novel drugs and treatment strategies be developed. Mouse models provide an important platform for anti-filarial drug research in a preclinical setting. This review presents an overview about the Litomosoides sigmodontis and Acanthocheilonema viteae filarial mouse models and their role in immunological research as well as preclinical studies about novel anti-filarial drugs and treatment strategies.

Filarial diseases like lymphatic filariasis and onchocerciasis belong to the Neglected Tropical Diseases and remain a public health problem in endemic countries. Lymphatic filariasis and onchocerciasis can lead to stigmatizing pathologies and present a socio-economic burden for affected people and their endemic countries. Current treatment recommendations by the WHO include mass drug administration with ivermectin for the treatment of onchocerciasis and a combination of ivermectin, albendazole and diethylcarbamazine (DEC) for the treatment of lymphatic filariasis in areas that are not co-endemic for onchocerciasis or loiasis. Limitations of these treatment strategies are due to potential severe adverse events in onchocerciasis and loiasis patients following DEC or ivermectin treatment, respectively, the lack of a macrofilaricidal efficacy of those drugs and the risk of drug resistance development. Thus, to achieve the elimination of transmission of onchocerciasis and the elimination of lymphatic filariasis as a public health problem by 2030, the WHO defined in its roadmap that new alternative treatment strategies with macrofilaricidal compounds are required. Within a collaboration of the non-profit organizations Drugs for Neglected Diseases initiative (DND

Filarial nematodes are parasitic worms transmitted by blood-feeding insects. Mainly found in tropical and subtropical areas of the developing world, diseases such as lymphatic filariasis and onchocerciasis represent major public health issues. With millions of people infected and billions at risk of infection, these diseases can stun economic growth and impair the life quality, hence the WHO classified both lymphatic filariasis and onchocerciasis as Neglected Tropical Diseases. The lesser known filarial disease loiasis is not only affecting millions of people, but represents a huge obstacle during mass drug administration programmes targeting other filarial diseases. Even less is known about mansonellosis, potentially the most widespread of the human filariases, but underestimated due to the lack of clinical symptoms. Large scale intervention as well as mass drug administration programmes are undertaken with the long term goal of eliminating the filarial diseases lymphatic filariasis and onchocerciasis. However, there is still neither a vaccination nor short term macrofilaricidal treatments available. The following chapter will encompass the different filarial diseases, the biology of the parasite and their vector, the epidemiology as well as pathology of the filariases, highlighting the impact of these diseases is still immense and further research in understanding and combating these diseases is needed.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα-/-/Il-5-/-) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα-/-/Il-5-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα-/-/Il-5-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80intermediate macrophages from infected Il-4rα-/-/Il-5-/- mice failed to mature into resident F4/80high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα-/-/Il-5-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

The range and burden of neglected tropical diseases, many of which are helminth-derived, remains enormous. Infections are rampant in poor districts and although efforts have been implemented over the years, there remains insufficient networks for disease control. In India, experts are encouraging the government to develop a functional public health infrastructure to manage diseases. India has 553 million people at risk for lymphatic filariasis (LF), one of the chronic filarial nematode infections that causes severe morbidity in humans. During coevolution, filariae have developed tactics to modulate the host’s immune system so that they can persist for many years. Therefore, most individuals remain asymptomatic and mansonellosis and loiasis are primarily thought of as nuisance infections. Nevertheless, pathology can develop into elephantiasis during LF and Onchocerca volvulus infections can lead to vision loss or skin pathology. Many filarial species require the endosymbiotic Wolbachia bacteria for development and maturation. Indeed, targeting Wolbachia via antibiotic therapy has provided an alternative therapeutic approach which, in contrast to drugs currently employed in mass drug administration programs, is highly macrofilaricidal. This chapter provides an overview about filarial agents drawing upon both their similarities and differences with regards to host immune reactions, ensuing pathologies and how infections alter response to vaccines and other diseases. All of these aspects have to be considered when implementing therapy, especially when adverse side effects may occur. These effects are synopsized in the final section alongside current success stories in terms of elimination and future strategies to control these public health problems.

Parasitic nematodes such as hookworms actively penetrate the skin of their hosts, encountering skin-resident innate immune cells that represent the host´s first line of defense. Here we useStrongyloides rattias a model for an intestinal helminth parasite with tissue migrating stages. We show that interception and killing of migrating larvae in mice during a 1stinfection occurred predominantly in skin and muscle tissue before larvae migratedvialung and head tissue to the intestine. Inhibition of larval migration was even more efficient in immune mice during a 2ndinfection where larvae barely left the site of entry i.e. the foot. Using cell-deficient mice we show that interception in the tissue was predominantly mediated by neutrophils and eosinophils while basophils and mast cells were dispensablein vivo. Likewise, neutrophils and eosinophils inhibitedS. rattiL3 motilityin vitroin the context of ETosis. Thereby eosinophils were strictly dependent on the presence of anti-S. rattiantibodies while neutrophils inhibited L3 motility as such. Also, MPO and MMP-9 were released by neutrophils in response to L3 alone, but immune plasma further stimulated MPO release in an antibody-dependent manner. In summary, our findings highlight the central role of the skin as first line of defense against helminth parasites in both, innate and adaptive immunity.

Summary Excessive inflammatory immune responses during infections with Plasmodium parasites are responsible for severe complications such as cerebral malaria (CM) that can be studied experimentally in mice. Dendritic cells (DCs) activate cytotoxic CD8+ T‐cells and initiate immune responses against the parasites. Batf3 −/− mice lack a DC subset, which efficiently induces strong CD8 T‐cell responses by cross‐presentation of exogenous antigens. Here we show that Batf3 −/− mice infected with Plasmodium berghei ANKA (PbA) were protected from experimental CM (ECM), characterized by a stable blood−brain barrier (BBB) and significantly less infiltrated peripheral immune cells in the brain. Importantly, the absence of ECM in Batf3 −/− mice correlated with attenuated responses of cytotoxic T‐cells, as their parasite‐specific lytic activity as well as the production of interferon gamma and granzyme B were significantly decreased. Remarkably, spleens of ECM‐protected Batf3 −/− mice had elevated levels of regulatory immune cells and interleukin 10. Thus, protection from ECM in PbA‐infected Batf3 −/− mice was associated with the absence of strong CD8+ T‐cell activity and induction of immunoregulatory mediators and cells., Batf3‐deficient mice lack cross‐presenting dendritic cells that are crucial for CD8 T‐cell activation. Here we show that these mice are protected from experimental cerebral malaria upon infection with Plasmodium berghei ANKA. The protected mice showed a decreased cytotoxic response and signs of immune regulation.

Microglia mediated responses to neuronal damage in the form of neuroinflammation is a common thread propagating neuropathology. In this study, we investigated the microglial alterations occurring as a result of sphingosine 1-phosphate (S1P) accumulation in neural cells. We evidenced increased microglial activation in the brains of neural S1P-lyase (SGPL1) ablated mice (SGPL1fl/fl/Nes ) as shown by an activated and deramified morphology and increased activation markers on microglia. In addition, an increase of pro-inflammatory cytokines in sorted and primary cultured microglia generated from SGPL1 deficient mice was noticed. Further, we assessed autophagy, one of the major mechanisms in the brain that keeps inflammation in check. Indeed, microglial inflammation was accompanied by defective microglial autophagy in SGPL1 ablated mice. Rescuing autophagy by treatment with rapamycin was sufficient to decrease interleukin 6 (IL-6) but not tumor necrosis factor (TNF) secretion in cultured microglia. Rapamycin mediated decrease of IL-6 secretion suggests a particular mechanistic target of rapamycin (mTOR)-IL-6 link and appeared to be microglia specific. Using pharmacological inhibitors of the major receptors of S1P expressed in the microglia, we identified S1P receptor 2 (S1PR2) as the mediator of both impaired autophagy and proinflammatory effects. In line with these results, the addition of exogenous S1P to BV2 microglial cells showed similar effects as those observed in the genetic knock out of SGPL1 in the neural cells. In summary, we show a novel role of the S1P-S1PR2 axis in the microglia of mice with neural-targeted SGPL1 ablation and in BV2 microglial cell line exogenously treated with S1P.

A type 1 immune response is involved in atherosclerosis progression, whereas the role of a type 2 polarization, especially with regard to an enhanced T helper (Th)2 cell differentiation, is still unclear. Helminths trigger type 2 immune responses, protecting the host from inflammatory disorders. We investigated whether an increased type 2 polarization by administration of Litomosoides sigmodontis adult worm extract (LsAg) affects atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Injections of 50 µg LsAg, i.p. into ApoE-/- mice induced a type 2 immune response shown by increased frequencies of peritoneal eosinophils and alternatively activated macrophages. To analyze the effect of LsAg on atherosclerosis initiation, ApoE-/- mice received a high-fat diet for 12 wk and weekly injections of 50 µg LsAg from wk 5 to 12. Therapeutic effects on advanced atherosclerosis were analyzed in mice that were fed a high-fat diet for 12 wk followed by 12 wk of normal chow and weekly LsAg injections. Both preventive and therapeutic LsAg application significantly decreased plaque size. Therapeutic treatment even caused regression of plaque size and macrophage density in the aortic root and reduced Th1-specific gene expression and intraplaque inflammation. In addition, plaque size after therapeutic treatment was inversely correlated with plaque-infiltrated alternatively activated macrophages. In vitro, LsAg treatment of HUVECs reduced intracellular levels of phosphorylated NF-κB-p65, IκB-α, and JNK1/2. In bifurcation flow-through slides, THP-1 cell adhesion to a HUVEC monolayer was decreased by LsAg in regions of nonuniform shear stress. Applying inhibitors of the respective kinases suggests JNK1/2 inhibition is involved in the suppressed cell adhesion. A switch to an enhanced type 2 immune response by LsAg exerts antiatherogenic effects on murine plaque development, indicating a protective role of a hampered type 1 polarization. In vitro, LsAg affects endothelial signaling pathways, among which JNK1/2 inhibition seems to be involved in the suppression of monocytic cell adhesion under proatherogenic shear stress.-Constanze, K., Tauchi, M., Furtmair, R., Urschel, K., Raaz-Schrauder, D., Neumann, A.-L., Frohberger, S. J., Hoerauf, A., Regus, S., Lang, W., Sagban, T. A., Stumpfe, F. M., Achenbach, S., Hubner, M. P., Dietel, B. Filarial extract of Litomosoides sigmodontis induces a type 2 immune response and attenuates plaque development in hyperlipidemic ApoE-knockout mice.

Introduction The subject of the present study was a systematic comparative analysis of the rheumatoid arthritis (RA)-induced pathomechanisms in the temporomandibular joint with those of the limb joints using the serum-induced arthritis K/BxN model. Methods In 18 BALB/c mice the induction of RA was performed according to the Kouskoff method. Another healthy cohort served as controls (n = 12). Joint swelling of the paws was measured using a micrometer. Functional data were obtained analyzing locomotion. Three-dimensional examination of the temporomandibular joint was performed with micro-computed tomography imaging, followed by histological evaluation of the extremity joints and the temporomandibular joint. Additionally, immunohistochemical investigations were carried out to evaluate inflammatory and immunological changes. Results Measurement of joint swelling showed a significant increase in the diameter of the paws, as well as a decrease in locomotor activity compared to control animals and the time before arthritis induction. Histological and immunohistochemical investigations showed clear signs of inflammation in the extremity joints. In contrast, no histological or immunohistochemical indications of an inflammatory process were detectable in the temporomandibular joint. In addition, the three-dimensional analysis by micro-computed tomography of the temporomandibular joints did not show any obvious morphological changes. Conclusion For the first time, using the K/BxN model we could demonstrate that, due to its anatomical and mechanical conditions, the temporomandibular joint seems to be less susceptible to the initiation of RA compared to limb joints. Therefore, additional investigations are needed on other arthritis models as well, in order to further improve our understanding of the pathogenesis and defense mechanisms of the disease.

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses toPlasmodiumparasites. Here we use a newly developed transgenicPlasmodium bergheiANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates thatIfnar1-/-mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protectedIfnar1-/-mice and wild type mice suffering from ECM. Importantly, CD8+T cells were increased in the spleens of ECM-protectedIfnar1-/-mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+T cell infiltration into the brain and increased ECM induction inPbAAma1OVA-infectedIfnar1-/-mice. However, eosinophil-depletion did not reduce the CD8+T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+T cell migration and proliferation duringPbAAma1OVA-infection inIfnar1-/-mice and thereby are contributing to the protection from ECM.

Background Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. Methodology/Principal findings Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems. Conclusions/Significance The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening., Author summary River blindness affects people living in mostly remote and underserved rural communities in some of the poorest areas of the world. Although significant efforts have been achieved towards the reduction of disease morbidity, onchocerciasis still affects millions of people in Sub-Saharan Africa. The current control strategy is the annual mass administration of ivermectin which has accumulated several drawbacks over time, especially the action of the drug is solely microfilaricidal, very long treatment period (15–17 years) and reports of ivermectin losing its efficacy; thus, raising the urgent need for new adulticidal compounds. Our study has established an in vitro platform capable of supporting the growth and development of Onchocerca volvulus for up to 315 days, enabling the observation of parasite developmental processes: moulting (from the infective L3 stage to adults), increase in morphometry, the attachment of adult male and female worms and the potential initiation of nodulogenesis. Moreover, the platform might provide more insight into O. volvulus adult worms behavioural pattern in vitro. Also, our findings provide more avenues for mass production of different parasite stages, the investigation of parasite developmental biology and the identification of targets for drug discovery against different developmental stages of this filarial parasite within 315 days.

Summary Eosinophils mediate protection against filarial nematodes. Our results demonstrate that eosinophil extracellular traps (EETosis) are induced by microfilariae and infective L3 larvae of Litomosoides sigmodontis. These extracellular DNA traps inhibit microfilariae motility in a DNA- and contact-dependent manner in vitro. Accordingly, microfilariae-injection triggers DNA release in an eosinophil-dependent manner in vivo and microfilariae covered with DNA traps are cleared more rapidly. Using dectin-1, we identify the required receptor for the microfilariae-induced EETosis, whereas signaling via other C-type lectin receptors, prior priming of eosinophils, and presence of antibodies are not required. The DNA released upon microfilariae-induced EETosis is mainly of mitochondrial origin, but acetylated and citrullinated histones are found within the traps. We further demonstrate that the presented DNA-dependent inhibition of microfilariae motility by eosinophils represents a conserved mechanism, as microfilariae from L. sigmodontis and the canine heartworm Dirofilaria immitis induce ETosis in murine and human eosinophils.

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer–DNDi partnership is an outstanding example of “One World Health,” in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area., Author summary Onchocerca volvulus is the causative agent of human river blindness, and current elimination programs rely on the use of ivermectin to kill microfilariae. Since no adulticidal drug is available and adult worms have a life span of up to 15 years, elimination programs need to be sustained over several decades. Emodepside is an anthelmintic that is licensed as a dewormer for cats and dogs. Due to its ability to eliminate nematodes located in various extraintestinal host tissues, including migrating larvae and adult filarial worms, it is considered to be an excellent candidate for the treatment of onchocerciasis. Intense collaboration between academia and the pharmaceutical industry has led to a deep understanding of the novel mode of action of the drug and of its parasite target spectrum. Phase I clinical trials with emodepside have demonstrated its safety and adulticide activity against the closely related cattle parasite Onchocerca ochengi. Currently, Phase II clinical trials are planned to confirm that emodepside, developed initially to improve animal health, has also the potential to improve human health by tackling a very important neglected tropical disease (NTD).