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Background Tumor-associated macrophages (TAMs) play a critical function in the development of tumors and are associated with protumor M2 phenotypes. Shifting TAMs towards antitumor M1 phenotypes holds promise for tumor immunotherapy. Oleamide, a primary fatty acid amide, has emerged as a potent anticancer and immunomodulatory compound. However, the regulatory effects of oleamide on TAM phenotypes remain unclear. Methods We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to study the influence of oleamide on primary human monocyte-derived TAM phenotypes, and we investigated the protein expression profiles based on mass spectrometry to analyze the effect of oleamide on macrophage polarization. Moreover, the advantageous binding scores between oleamide and these target candidate proteins are examined using molecular docking. Results Our study revealed that oleamide effectively suppressed the M2-like TAM phenotype by reducing interleukin (IL)-10 production and downregulating M2-like markers, including vascular endothelial growth factor A (VEGFA), MYC proto-oncogene, bHLH transcription factor (c-Myc), and mannose receptor C-type 1 (CD206). Moreover, the conditioned medium derived from oleamide-treated TAMs induces apoptosis of MDA-MB-231 breast cancer cells. Proteomic analysis identified 20 candidate up- and down-regulation proteins targeted by oleamide, showing modulation activity associated with the promotion of the M1-like phenotype. Furthermore, molecular docking demonstrated favorable binding scores between oleamide and these candidate proteins. Collectively, our findings suggest that oleamide exerts a potent antitumor effect by promoting the antitumor M1-like TAM phenotype. These novel insights provide valuable resources for further investigations into oleamide and macrophage polarization which inhibit the progression of breast cancer, which may provide insight into immunotherapeutic approaches for cancer.

Summary: Precise, on-target CRISPR-Cas9 genome editing has been shown in Schistosoma mansoni, involving both non-homology end joining and homology-directed repair pathways. Here, we present a multiplexed CRISPR-Cas9 protocol for large transgene integration into the S. mansoni genome. We describe steps for deploying multiplexed ribonucleoprotein complexes (RNPs) and donor DNA preparation. We then detail procedures for introducing RNPs into schistosome eggs by square-wave electroporation in the presence of a 5′ phosphorothioate-modified double-stranded donor transgene.For complete details on the use and execution of this protocol, please refer to Ittiprasert et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Background: Opisthorchiasis is caused by an infection with fish-borne liver flukes of the genus Opisthorchis. Opisthorchiasis frequently leads to chronic inflammation in the biliary tract and is classified as a group 1 biological carcinogen by the International Agency for Research on Cancer: a definitive risk for cholangiocarcinoma (CCA). Methods: We used the rapid immunochromatographic test (ICT) to detect anti-Opisthorchis viverrini IgG and IgG4 subclass antibodies in sera of patients with CCA. The ICT kits were developed based on soluble antigens excreted and secreted by O. viverrini adult worms. Results: ICT indicated sera was positive for IgG and IgG4 antibodies, respectively, in 22 (61.1%) and 15 (41.6%) participants of the 36 study participants diagnosed with CCA (P > 0.05). Our study also included groups with other cancers and with liver cirrhosis, where the IgG ICT and IgG4 ICT kits were 27.7% (13/47) and 25.5% (12/47) positive, respectively (P > 0.05). Neither total the IgG ICT nor the IgG4 ICT yielded positive results in a control group of 20 healthy participants. Moreover, the percentage positivity rate using the ICT for total IgG between the CCA group and the other cancers and liver cirrhosis group was significantly different (P < 0.05). By contrast, no significant difference between these groups was apparent in the ICT for IgG4 antibody. The CCA group was 6.53 times more likely to have positive anti–O. viverrini IgG antibody (odds ratio 6.53, P < 0.001) and 3.27 times more likely to have positive anti–O. viverrini IgG4 antibody (odds ratio 3.27, P = 0.010) than the non-CCA group. Conclusion: This information is of potential value for the development of a diagnostic biomarker to predict risk for O. viverrini infection-associated CCA.

Abstract Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross‐species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well‐recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth‐derived EVs in these processes and highlights EVs as an important participant in cross‐phylum communication. While the mammalian EV field is guided by a community‐agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan‐helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth‐derived EVs to complement the MISEV guidelines. We summarise community‐agreed standards for studying EVs derived from this broad set of non‐model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Abstract Background Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted “infiltrin” protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE’s effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium. Summary Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium.

Abstract Background Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. Methods Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). Conclusions The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition. Keywords: Opisthorchis viverrini, Liver fluke granulin, Extracellular vesicle, Cellular crosstalk

Inter-phylum transfer of molecular information is exquisitely exemplified in the uptake of parasite extracellular vesicles (EVs) by their target mammalian host tissues. The oriental liver fluke, Opisthorchis viverrini is the major cause of bile duct cancer in people in Southeast Asia. A major mechanism by which O. viverrini promotes cancer is through the secretion of excretory/secretory products which contain extracellular vesicles (OvEVs). OvEVs contain microRNAs that are predicted to impact various mammalian cell proliferation pathways, and are internalized by cholangiocytes that line the bile ducts. Upon uptake, OvEVs drive relentless proliferation of cholangiocytes and promote a tumorigenic environment, but the underlying mechanisms of this process are unknown. Moreover, purification and characterization methods for helminth EVs in general are ill defined. We therefore compared different purification methods for OvEVs and characterized the sub-vesicular compartment proteomes. Two CD63-like tetraspanins (Ov-TSP-2 and TSP-3) are abundant on the surface of OvEVs, and could serve as biomarkers for these parasite vesicles. Anti-TSP-2 and -TSP-3 IgG, as well as different endocytosis pathway inhibitors significantly reduced OvEV uptake and subsequent proliferation of cholangiocytes in vitro. Silencing of Ov-tsp-2 and tsp-3 gene expression in adult flukes using RNA interference resulted in substantial reductions in OvEV secretion, and those vesicles that were secreted were deficient in their respective TSP proteins. Our findings shed light on the importance of tetraspanins in fluke EV biogenesis and/or stability, and provide a conceivable mechanism for the efficacy of anti-tetraspanin subunit vaccines against a range of parasitic helminth infections.

Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover ‘new’ genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic ‘signatures’ that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis. Author summary More than 100 million people are infected with the carcinogenic blood fluke Schistosoma haematobium, the aetiological agent of urogenital schistosomiasis—a neglected tropical disease (NTD). In spite of its major significance, little is known about this fluke, its interactions with the human and snail intermediate hosts and the pathogenesis of the urogenital form of schistosomiasis at the molecular and biochemical levels. To enable research in these areas, we report the first chromosome-level genome and markedly enhanced gene models for S. haematobium. Comparative genomic analyses also reveal evidence of past introgression events between or among closely related schistosome species. This present reference genome for S. haematobium and the findings from this study should underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved control of urogenital schistosomiasis.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm’s excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

Chronic human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis can last for decades and cause liver and biliary diseases, including life-threatening pathology prior to cholangiocarcinoma (CCA). CCA generally has a poor prognosis. Serological diagnosis can support parasitological examination in diagnosing disease and screening for the risk of CCA. Here, we present an improved and innovative lateral flow immunochromatographic test (ICT) kit that uses whole-blood samples (WBS) rather than serum to diagnose human opisthorchiasis, which also successfully diagnosed human clonorchiasis. This ICT includes a soluble worm extract of O. viverrini adults and colloidal-gold-labeled conjugates of the IgG antibody to evaluate the diagnostic values with simulated WBS (n = 347). Simulated WBS were obtained by the spiking infection sera with red blood cells. The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy for detecting opisthorchiasis were 95.5%, 87.0%, 80.5%, 97.2%, and 90.1%, respectively. For clonorchiasis, these findings were 85.7%, 87.0%, 53.6%, 97.2%, and 86.8%, respectively. Combined for both diseases, they were 93.2%, 87.0%, 84.0%, 94.6%, and 89.6%, respectively. The ICT kit can possibly replace the ICT platforms for antibody detection in serum samples in field surveys in remote areas where sophisticated equipment is not available.

Abstract Background Infections classified as group 1 biological carcinogens include the helminthiases caused by Schistosoma haematobium and Opisthorchis viverrini. The molecular mediators underlying the infection with these parasites and cancer remain unclear. Although carcinogenesis is a multistep process, we have postulated that these parasites release metabolites including oxysterols and estrogen-like metabolites that interact with host cell DNA. How and why the parasite produce/excrete these metabolites remain unclear. A gene encoding a CYP enzyme was identified in schistosomes and opisthorchiids. Therefore, it is reasonable hypothesized that CYP 450 might play a role in generation of pro-inflammatory and potentially carcinogenic compounds produced by helminth parasites such as oxysterols and catechol estrogens. Here, we performed enzymatic assays using several isoforms of CYP 450 as CYP1A1, 2E1 and 3A4 which are involved in the metabolism of chemical carcinogens that have been associated with several cancer. The main aim was the analysis of the role of these enzymes in production of helminth-associated metabolites and DNA-adducts. Method The effect of cytochrome P450 enzymes CYP 1A1, 2E1 and 3A4 during the interaction between DNA, glycocholic acid and taurochenodeoxycholate sodium on the formation of DNA-adducts and metabolites associated with urogenital schistosomiasis (UGS) and opisthorchiasis was investigated in vitro. Liquid chromatography/mass spectrometry was used to detect and identify metabolites. Main findings Through the enzymatic assays we provide a deeper understanding of how metabolites derived from helminths are formed and the influence of CYP 450. The assays using compounds similar to those previously observed in helminths as glycocholic acid and taurochenodeoxycholate sodium, allowed the detection of metabolites in their oxidized form and their with DNA. Remarkably, these metabolites were previously associated with schistosomiaisis and opisthorchiasis. Thus, in the future, it may be possible to synthesize this type of metabolites through this methodology and use them in cell lines to clarify the carcinogenesis process associated with these diseases. Principal conclusions Metabolites similar to those detected in helminths are able to interact with DNA in vitro leading to the formation of DNA adducts. These evidences supported the previous postulate that imply helminth-like metabolites as initiators of helminthiases-associated carcinogenesis. Nonetheless, studies including these kinds of metabolites and cell lines in order to evaluate its potential carcinogenic are required.

Abstract Background Treatment of schistosomiasis has relied on the anthelmintic drug praziquantel (PZQ) for more than a generation. Despite its celebrated performance for treatment and control of schistosomiasis and other platyhelminth infections, praziquantel has some shortcomings and the inability of this drug to counteract disease sequelae prompts the need for novel therapeutic strategies. Methods Using a host-parasite model involving Biomphalaria glabrata and Schistosoma mansoni we established mechanical transformation of S. mansoni cercariae into newly transformed schistosomula (NTS) and characterized optimal culture conditions. Thereafter, we investigated the antischistosomal activity and ability of the antioxidants N-acetylcysteine (NAC) and resveratrol (RESV) to augment the performance of praziquantel and/or artesunate (AS) against larval stages of the parasite. Drug effects were evaluated by using an automated microscopical system to study live and fixed parasites and by transmission electron microscopy (TEM). Results Transformation rates of cercariae to schistosomula reached ~ 70% when the manipulation process was optimized. Several culture media were tested, with M199 supplemented with HEPES found to be suitable for S. mansoni NTS. Among the antioxidants studied, RESV alone or combined with anthelminthic drugs achieved better results rather N-acetylcysteine (NAC). TEM observations demonstrated that the combination of AS + RESV induced severe, extensive alterations to the tegument and subtegument of NTS when compared to the constituent compounds alone. Two anthelmintic–antioxidant combinations, praziquantel-resveratrol [combination index (CI) = 0.74] and artesunate-resveratrol (CI = 0.34) displayed moderate and strong synergy, respectively. Conclusions The use of viability markers including staining with propidium iodide increased the accuracy of drug screening assays against S. mansoni NTS. The synergies observed might be the consequence of increased action by RESV on targets of AS and PZQ and/or they may act through concomitantly on discrete targets to enhance overall antischistosomal action. Combinations of active agents, preferably with discrete modes of action including activity against developmental stages and/or the potential to ameliorate infection-associated pathology, might be pursued in order to identify novel therapeutic interventions.

Abstract Background The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. Methods The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were ‘spiked’ with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. Results In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi, 15 as Dirofilaria immitis and 7 as| D. repens. Conclusions We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.

Helminthiases are extremely prevalent in the developing world. In addition, the chronic infection with some parasitic worms are classified as carcinogenic. Therefore, it is utmost importance to understand the parasite-host interactions, the mechanisms underlay carcinogenesis and how they could be counteracted. This knowledge may ultimately guide novel control strategies that include chemotherapy-based approaches targeting these pathogens and associated pathologies caused by their infections. Little is known on how some helminthiases are associated with cancer; however, it has been hypothesized that chemical carcinogenesis may be involved in the process. Here, we summarize the current knowledge on chemical carcinogenesis associated with helminthiases, along with available therapeutic options and potential therapeutic alternatives including chemotherapy and/or immunotherapy. Ideally, the treatment of the carcinogenic helminthiases should target both the parasite and associated pathologies. The success of any chemotherapeutic regimen often depends on the host immune response during the infection and nutritional status among other factors. The close association between chemotherapy and cell-mediated immunity suggests that a dual therapeutic approach would be advantageous. In addition, there is a pressing need for complementary drugs that antagonize the carcinogenesis process associated with the helminth infections.

Earlier reports revealed oxysterol metabolites of Opisthorchis spp. liver fluke origin conjugated with DNA bases, suggesting that the generation of these DNA-adducts may underlie the mutagenicity and carcinogenicity of the infection with these food-borne pathogens. Here, we employed liquid chromatography-mass spectrometry to investigate, compare and contrast spectrograms of soluble extracts from Fasciola hepatica adult worms from bile ducts of cattle with those from O. viverrini and O.felineus from experimentally infected hamsters. F. hepatica and Opisthorchis spp. shared common compounds including oxysterol-like metabolites, bile acids and DNA-adducts, but the spectrometric profiles of F. hepatica included far fewer compounds than Opisthorchis species. These findings support the postulate that parasitic oxysterol-like metabolites could be related to carcinogenesis associated to infection and they point to a molecular basis for the differences among major groups of liver flukes concerning infection-induced malignancy.

Schistosomiasis remains a major neglected tropical disease. The treatment and control of schistosomiasis rely on a single drug, praziquantel (PZQ). Despite its efficacy, treatment with PZQ presents some major drawbacks including an inability of the chemotherapy to reverse disease-induced fibrosis and the prospect of the emergence of drug resistance. Here, we investigated a novel therapeutic approach with antioxidant biomolecules in combination with PZQ against the adult developmental stage of Schistosoma mansoni and oviposition in vitro, given that this therapeutic approach achieved synergistic/additive activity against larval schistosomes. The antioxidants curcumin and oxadiazole per se exhibited antischistosomal activity against adult worms leading to severe morphological alterations and death. Additionally, the antioxidant flavone combined with vandetanib or imatinib improved antischistosomal activity against adult forms. By contrast, however, these antioxidant-anthelmintic combinations were not as effective against adults in comparison to larval schistosomes. Nevertheless, the antioxidants alone or combined with drugs inhibited oviposition.

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

Opisthorchis viverrini causes severe pathology in the bile ducts of infected human hosts, and chronic infection can culminate in bile duct cancer. The prevention of infection by vaccination would decrease opisthorchiasis-induced morbidity and mortality. The tetraspanin protein, Ov-TSP-2, is located on the membrane of secreted extracellular vesicles (EVs), and is a candidate antigen for inclusion in a subunit vaccine. To address the role of anti-Ov-TSP-2 antibodies in protection, we assessed the protective capacity of anti-Ov-TSP-2 monoclonal antibodies (mAbs) against opisthorchiasis. Two anti-TSP-2 IgM mAbs, 1D6 and 3F5, and an isotype control were passively transferred to hamsters, followed by parasite challenge one day later. Hamsters that received 3F5 had 74.5% fewer adult flukes and 67.4% fewer eggs per gram of feces compared to hamsters that received the control IgM. Both 1D6 and 3F5 (but not the control IgM) blocked the uptake of fluke EVs by human bile duct epithelial cells in vitro. This is the first report of passive immunization against human liver fluke infection, and the findings portend the feasibility of antibody-directed therapies for liver fluke infection, bolstering the selection of TSPs as components of a subunit vaccine for opisthorchiasis and fluke infections generally.

Cancer may be induced by many environmental and physiological conditions. Infections with viruses, bacteria and parasites have been recognized for years to be associated with human carcinogenicity. Here we review current concepts of carcinogenicity and its associations with parasitic infections. The helminth diseases schistosomiasis, opisthorchiasis, and clonorchiasis are highly carcinogenic while the protozoan Trypanosoma cruzi, the causing agent of Chagas disease, has a dual role in the development of cancer, including both carcinogenic and anticancer properties. Although malaria per se does not appear to be causative in carcinogenesis, it is strongly associated with the occurrence of endemic Burkitt lymphoma in areas holoendemic for malaria. The initiation of Plasmodium falciparum related endemic Burkitt lymphoma requires additional transforming events induced by the Epstein-Barr virus. Observations suggest that Strongyloides stercoralis may be a relevant co-factor in HTLV-1-related T cell lymphomas. This review provides an overview of the mechanisms of parasitic infection-induced carcinogenicity.

Aims: There is a general, inverse relationship between helminth infection and allergic diseases including bronchial asthma (BA). Proteins and other mediators released from parasitic worms exert cogent downmodulation of atopic and other allergic reactivity. We investigated the immune activities of an immortalized murine dendritic cell (mDC) line (JAWSII) and of primary human dendritic cells (hDCs) collected from study participants with and without BA after Opisthorchis felineus hemozoin (OfHz) treatment.Methods and Results:in vitro, expression of lymphocyte-activating factors—T helper 1 (Th1) induction and anti-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), IL-10, and IL-12β–increased significantly in mDCs pulsed with OfHz. In parallel, primary dendritic cells (hDC) from cases clinically diagnosed with BA along with healthy controls were exposed ex vivo to OfHz in combination with lipopolysaccharide (LPS). Whereas no significant change in the cellular maturation markers, CD83, CD86, and CD40, was apparent in BA vs. healthy hDC, pulsing hDC from BA with OfHz with LPS induced significant increases in expression of IL-10 and IL-12β, although not of TNF-α or tumor growth factor-beta (TGF-β).Conclusions: Liver fluke hemozoin OfHz stimulated production of Th1 inducer and anti-inflammatory cytokines IL-10 and IL-12β from BA-hDC pulsed with OfHz, an outcome that enhances our understanding of the mechanisms whereby opisthorchiasis contributes to protection against the atopic disease in liver fluke infection-endemic regions.

Recent reports suggest that the East Asian liver fluke infection, caused by Opisthorchis viverrini, which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the cell of origin of this malignancy. Here, we investigated interactions in vitro among human cholangiocytes, Helicobacter pylori strain NCTC 11637, and the congeneric bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte of the H69 cell line induced phenotypic changes including the profusion of thread-like filopodia and a loss of cell-cell contact, in a dose-dependent fashion. In parallel, following exposure to H. pylori, changes were evident in levels of mRNA expression of epithelial to mesenchymal transition (EMT)-encoding factors including snail, slug, vimentin, matrix metalloprotease, zinc finger E-box-binding homeobox, and the cancer stem cell marker CD44. Analysis to quantify cellular proliferation, migration, and invasion in real-time by both H69 cholangiocytes and CC-LP-1 line of cholangiocarcinoma cells using the xCELLigence approach and Matrigel matrix revealed that exposure to ≥10 H. pylori bacilli per cell stimulated migration and invasion by the cholangiocytes. In addition, 10 bacilli of H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection by H.pylori contributes to the malignant transformation of the biliary epithelium.

The liver fluke Opisthorchis viverrini is a food-borne, zoonotic pathogen endemic to Thailand and adjacent countries in Southeast Asia. The adult developmental stage of the O. viverrini parasite excretes and secretes numerous proteins within the biliary tract including the gall bladder. Lesions caused by the feeding activities of the liver fluke represent wounds that undergo protracted cycles of healing and re-injury during chronic infection, which can last for decades. Components of the excretory/secretory (ES) complement released by the worms capably drive proliferation of bile duct epithelial cells and are implicated in establishing the oncogenic milieu that leads to bile duct cancer, cholangiocarcinoma. An ES protein, the secreted granulin-like growth factor termed Ov-GRN-1, accelerates wound resolution in mice and in vitro. To investigate angiogenesis (blood vessel development) that may contribute to wound healing promoted by liver fluke granulin and, by implication, to carcinogenesis during chronic opisthorchiasis, we employed an in vitro tubule formation assay (TFA) where human umbilical vein endothelial cells were grown on gelled basement matrix. Ten and 40 nM Ov-GRN-1 significantly stimulated angiogenesis as monitored by cellular proliferation and by TFA in real time. This demonstration of potent angiogenic property of Ov-GRN-1 bolsters earlier reports on the therapeutic potential for chronic non-healing wounds of diabetics, tobacco users, and the elderly and, in addition, showcases another of the hallmark of cancer characteristic of this carcinogenic liver fluke.

Infection with helminth parasites causes morbidity and mortality in billions of people and livestock worldwide. Where anthelmintic drugs are available, drug resistance is a major problem in livestock parasites, and a looming threat to public health. Monitoring the efficacy of these medicines and screening for new drugs has been hindered by the lack of objective, high-throughput approaches. Several cell monitoring technologies have been adapted for parasitic worms, including video-, fluorescence-, metabolism enzyme- and impedance-based tools that minimize the screening bottleneck. Using the xCELLigence impedance-based system we previously developed a motility-viability assay that is applicable for a range of helminth parasites. Here we have improved substantially the assay by using diverse frequency settings, and have named it the xCELLigence worm real-time motility assay (xWORM). By utilizing strictly standardized mean difference analysis we compared the xWORM output measured with 10, 25 and 50 kHz frequencies to quantify the motility of schistosome adults (human blood flukes) and hatching of schistosome eggs. Furthermore, we have described a novel application of xWORM to monitor movement of schistosome cercariae, the developmental stage that is infectious to humans. For all three stages, 25 kHz was either optimal or near-optimal for monitoring and quantifying schistosome motility. These improvements in methodology sensitivity should enhance the capacity to screen small compound libraries for new drugs both for schistosomes and other helminth pathogens at large.

ABSTRACT Understanding how microbiomes affect host resistance, parasite virulence, and parasite-associated diseases requires a collaborative effort between parasitologists, microbial ecologists, virologists, and immunologists. We hereby propose the Parasite Microbiome Project to bring together researchers with complementary expertise and to study the role of microbes in host-parasite interactions. Data from the Parasite Microbiome Project will help identify the mechanisms driving microbiome variation in parasites and infected hosts and how that variation is associated with the ecology and evolution of parasites and their disease outcomes. This is a call to arms to prevent fragmented research endeavors, encourage best practices in experimental approaches, and allow reliable comparative analyses across model systems. It is also an invitation to foundations and national funding agencies to propel the field of parasitology into the microbiome/metagenomic era.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer, arising in the biliary ducts that extend into the liver. The highest incidence of ICC occurs in Southeast Asia, particularly in the Mekong River Basin countries of Thailand, Laos, Cambodia, and Vietnam, where it is strongly associated with chronic infection by the food-borne liver fluke Opisthorchis viverrini (OV), one of only three eukaryote pathogens considered Group one carcinogens. Intrahepatic cholangiocarcinoma is usually diagnosed at an advanced stage, with a poor prognosis and survival often less than 24 months. Hence, biomarkers that enable the early detection of ICC would be desirable and have a potentially important impact on the public health in the resource-poor regions where this cancer is most prevalent. As microRNAs (miRNAs) remain well preserved after formalin fixation, there is much interest in developing them as biomarkers that can be investigated using tumor biopsy samples preserved in formalin fixed paraffin embedded (FFPE) tumor blocks. Recently, we reported the first comprehensive profiling of tissue-based miRNA expression using FFPE from the three most common subtypes of OV-induced ICC tumors: moderately differentiated ICC, papillary ICC, and well-differentiated ICC. We observed that each subtype of OV-induced ICC exhibited a distinct miRNA profile, which suggested the involvement of specific sets of miRNAs in the progression of this cancer. In addition, non-tumor tissue adjacent to ICC tumor tissue on the same FFPE block shared a similar miRNA dysregulation profile with the tumor tissue than with normal (non-tumor) liver tissue (individuals without ICC or OV infection). Herein, we provide a detailed description of the microarray analysis procedures used to derive these findings.

Nasopharyngeal carcinoma (NPC) is a non-lymphomatous, squamous-cell carcinoma that occurs in the epithelial lining of the nasopharynx. Nasopharyngeal carcinoma has a geographically well-defined distribution worldwide, with the highest prevalence in China, Southeast Asia, and Northern Africa. Symptoms of nascent NPC may be unapparent or trivial, with diagnosis based on the histopathology of biopsied tissue following endoscopy of the nasopharynx. The tumor node metastasis (TNM) staging system is the benchmark for the prognosis of NPC and guides treatment strategy. However, there is a consensus that the TNM system is not sufficiently specific for the prognosis of NPC, as it does not reflect the biological heterogeneity of this tumor, making another biomarker for the detection of NPC a priority. We have previously reported on different approaches for microRNA (miRNA) biomarker discovery for Formalin Fixed Paraffin Embedded (FFPE) NPC tissue samples by both a targeted (microarray) and an untargeted (small RNA-Seq) discovery platform. Both miRNA discovery platforms produced similar results, narrowing the miRNA signature to 1–5% of the known mature human miRNAs, with untargeted (small RNA-Seq approach) having the advantage of indicating “unknown” miRNAs associated with NPC. Both miRNA profiles strongly associated with NPC, providing two potential discovery platforms for biomarker signatures for NPC. Herein, we provide a detailed description of the methods that we used to interrogate FFPE samples to discover biomarkers for NPC.

Schistosomiasis is a major neglected tropical disease. Treatment for schistosomiasis with praziquantel (PZQ), which is effective against the parasite, by itself is not capable to counteract infection-associated disease lesions including hepatic fibrosis. There is a pressing need for novel therapies. Due to their biological properties, antioxidant biomolecules might be useful in treating and reverting associated pathological sequelae. Here, we investigated a novel therapy approach based on a combination of anthelmintic drugs with antioxidant biomolecules. We used a host-parasite model involving Bioamphalaria glabrata and newly transformed schistosomula (NTS) of Schistosoma mansoni. For in vitro drug screening assays, was selected several antioxidants and evaluated not only antischistosomal activity but also ability to enhance activity of the anthelmintic drugs praziquantel (PZQ) and artesunate (AS). The morphological alterations induced by compounds alone/combined were assessed on daily basis using an inverted and automated microscope to quantify NTS viability by a fluorometric-based method. The findings indicated that not only do some antioxidants improve antischistosomal activity of the two anthelmintics, but they exhibit activity per se, leading to high mortality of NTS post-exposure. The combination index (CI) of PZQ + Mel (CI = 0.80), PZQ + Resv (CI = 0.74), AS + Resv (CI = 0.34), AS + NAC (CI = 0.89), VDT + Flav (CI = 1.03) and VDT + Resv (CI = 1.06) reveal that they display moderate to strong synergism. The combination of compounds with discrete mechanisms of action might provide a valuable adjunct to contribution for treatment of schistosomiasis-associated disease.

Schistosomiasis is a major neglected tropical disease. Control of schistosomiasis currently relies on a single drug, praziquantel, and despite its efficacy against the all schistosome species that parasitize humans, it displays some problematic drawbacks and alone is ineffective in counteracting adverse pathologies associated with infection. Moreover, due to the development of the potential emergence of PZQ-resistant strains, the search for additional or alternative antischistosomal drugs have become a public health priority. The current drug discovery for schistosomiasis has been slow and uninspiring. By contrast, repurposing of existing approved drugs may offer a safe, rapid and cost-effective alternative. Combined treatment with PZQ and other drugs with different mode of action, i.e., antimalarials, shows promise results. In addition, a combination of anthelminthic drugs with antioxidant might be advantageous for modulating oxidative processes associated with schistosomiasis. Herein, we review studies dealing with combination therapies that involve PZQ and other anthelminthic drugs and/or antioxidant agents in treatment of schistosomiasis. Whereas PZQ combined with antioxidant agents might or might not interfere with anthelminthic efficacy, combinations may nonetheless ameliorate tissue damage and infection-associated complications. In fact, alone or combine with other drugs, antioxidants might be a valuable adjuvant to reduce morbidity and mortality of schistosomiasis. Therefore, attempting new combinations of anthelmintic drugs with other biomolecules such as antioxidants provides new avenues for discovery of alternatives to PZQ.

This paper reports recent observations from our laboratory dealing with the anti-schistosome drugs hycanthone (HC) and praziquantel (PZQ). In particular, we discuss a laboratory model of drug resistance to HC in Schistosoma mansoni and show that drug sensitive and resistant lines of the parasite can be differentiated on the basis of restriction fragment length polymorphisms using homologous ribosomal gene probes. In addition, we summarize data demonstrating that effective chemotherapy of S. mansoni infection with PZQ in mice requires the presence of host anti-parasite antibodies. These antibodies bind to PZQ treated worms and may be involved in an antibody-dependent cellular cytotoxicity reactions which result in the clearance of worms from the vasculature.