European propolis is highly active against trypanosomatids including Crithidia fasciculata

Extracts of 35 samples of European propolis were tested against wild type and resistant strains of the protozoal pathogens Trypanosoma brucei, Trypanosoma congolense and Leishmania mexicana. The extracts were also tested against Crithidia fasciculata a close relative of Crithidia mellificae, a parasite of bees. Crithidia, Trypanosoma and Leishmania are all members of the order Kinetoplastida. High levels of activity were obtained for all the samples with the levels of activity varying across the sample set. The highest levels of activity were found against L. mexicana. The propolis samples were profiled by using liquid chromatography with high resolution mass spectrometry (LC-MS) and principal components analysis (PCA) of the data obtained indicated there was a wide variation in the composition of the propolis samples. Orthogonal partial least squares (OPLS) associated a butyrate ester of pinobanksin with high activity against T. brucei whereas in the case of T. congolense high activity was associated with methyl ethers of chrysin and pinobanksin. In the case of C. fasciculata highest activity was associated with methyl ethers of galangin and pinobanksin. OPLS modelling of the activities against L. mexicana using the mass spectrometry produced a less successful model suggesting a wider range of active components

Protection from T cell-dependent colitis by the helminth-derived immunomodulatory mimic of transforming growth factor-ß, Hp-TGM

In animal models of inflammatory colitis, pathology can be ameliorated by several intestinal helminth parasites, including the mouse nematode Heligmosomoides polygyrus. To identify parasite products that may exert anti-inflammatory effects in vivo, we tested H. polygyrus excretory-secretory (HES) products, as well as a recombinantly-expressed parasite protein, TGM, that functionally mimics the mammalian immunomodulatory cytokine TGF-β. HES and TGM showed a degree of protection in DSS-induced colitis, with a reduction in inflammatory cytokines, but did not fully block the development of pathology. HES also showed little benefit in a similar acute TNBS-induced model. However, in a T cell transfer-mediated model with RAG-deficient mice, HES reduced disease scores if administered throughout the first 2 or 4 weeks following transfer but was less effective if treatment was delayed until 14 days after T cell transfer. Recombinant TGM similarly dampened colitis in RAG-deficient recipients of effector T cells, and was effective even if introduced only once symptoms had begun to be manifest. These results are a promising indication that TGM may replicate, and even surpass, the modulatory properties of native parasite HES

Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross‐resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogues DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3H]‐diminazene was slow, low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene‐resistant T. b. brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3H]‐diminazene transport studies, whole genome sequencing and RNA‐seq found no major changes in diminazene uptake, folate transporter sequence or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance

Cloning and functional complementation of ten <i>Schistosoma mansoni</i> phosphodiesterases expressed in the mammalian host stages

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals

Ethnopharmacological relevance: Leaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. Aim of the study: To investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. Material and methods: Plants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. Results: Seven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38 µg/mL, while a triterpenoid (3β,13β-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 of 1.58 µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400 µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. Conclusion: We have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species

Suppression of airway allergic eosinophilia by Hp-TGM, a helminth mimic of TGF-β

Type 2-high asthma is a chronic inflammatory disease of the airways which is increasingly prevalent in countries where helminth parasite infections are rare, and characterized by T helper 2 (Th2)-dependent accumulation of eosinophils in the lungs. Regulatory cytokines such as TGF-β can restrain inflammatory reactions, dampen allergic Th2 responses, and control eosinophil activation. The murine helminth parasite Heligmosomoides polygyrus releases a TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β despite bearing no structural similarity to the mammalian protein. Here, we investigated if Hp-TGM could alleviate allergic airway inflammation in mice exposed to Alternaria alternata allergen, house dust mite (HDM) extract or alum-adjuvanted ovalbumin protein (OVA). Intranasal administration of Hp-TGM during Alternaria exposure sharply reduced airway and lung tissue eosinophilia along with bronchoalveolar lavage fluid IL-5 and lung IL-33 cytokine levels at 24 hours. The protective effect of Hp-TGM on airway eosinophilia was also obtained in the longer T-cell mediated models of HDM or OVA sensitisation with significant inhibition of eotaxin-1, IL-4 and IL-13 responses depending on the model and time-point. Hp-TGM was also protective when administered parenterally either when given at the time of allergic sensitisation or during airway allergen challenge. This project has taken the first steps in identifying the role of Hp-TGM in allergic asthma and highlighted its ability to control lung inflammation and allergic pathology. Future research will investigate the mode of action of Hp-TGM against airway allergic eosinophilia, and further explore its potential to be developed as a biotherapeutic in allergic asthma