Near-infrared spectroscopy and machine learning algorithms for rapid and non-invasive detection of Trichuris.

BACKGROUND: Trichuris trichiura (whipworm) is one of the most prevalent soil transmitted helminths (STH) affecting 604-795 million people worldwide. Diagnostic tools that are affordable and rapid are required for detecting STH. Here, we assessed the performance of the near-infrared spectroscopy (NIRS) technique coupled with machine learning algorithms to detect Trichuris muris in faecal, blood, serum samples and non-invasively through the skin of mice. METHODOLOGY: We orally infected 10 mice with 30 T. muris eggs (low dose group), 10 mice with 200 eggs (high dose group) and 10 mice were used as the control group. Using the NIRS technique, we scanned faecal, serum, whole blood samples and mice non-invasively through their skin over a period of 6 weeks post infection. Using artificial neural networks (ANN) and spectra of faecal, serum, blood and non-invasive scans from one experiment, we developed 4 algorithms to differentiate infected from uninfected mice. These models were validated on mice from a second independent experiment. PRINCIPAL FINDINGS: NIRS and ANN differentiated mice into the three groups as early as 2 weeks post infection regardless of the sample used. These results correlated with those from concomitant serological and parasitological investigations. SIGNIFICANCE: To our knowledge, this is the first study to demonstrate the potential of NIRS as a diagnostic tool for human STH infections. The technique could be further developed for large scale surveillance of soil transmitted helminths in human populations.

Effect of experimental hookworm infection on insulin resistance in people at risk of type 2 diabetes.

The reduced prevalence of insulin resistance and type 2 diabetes in countries with endemic parasitic worm infections suggests a protective role for worms against metabolic disorders, however clinical evidence has been non-existent. This 2-year randomised, double-blinded clinical trial in Australia of hookworm infection in 40 male and female adults at risk of type 2 diabetes assessed the safety and potential metabolic benefits of treatment with either 20 (n = 14) or 40 (n = 13) Necator americanus larvae (L3) or Placebo (n = 13) (Registration ACTRN12617000818336). Primary outcome was safety defined by adverse events and completion rate. Homoeostatic model assessment of insulin resistance, fasting blood glucose and body mass were key secondary outcomes. Adverse events were more frequent in hookworm-treated participants, where 44% experienced expected gastrointestinal symptoms, but completion rates were comparable to Placebo. Fasting glucose and insulin resistance were lowered in both hookworm-treated groups at 1 year, and body mass was reduced after L3-20 treatment at 2 years. This study suggests hookworm infection is safe in people at risk of type 2 diabetes and associated with improved insulin resistance, warranting further exploration of the benefits of hookworms on metabolic health.

Peptides derived from hookworm anti-inflammatory proteins suppress inducible colitis in mice and inflammatory cytokine production by human cells.

A decline in the prevalence of parasites such as hookworms appears to be correlated with the rise in non-communicable inflammatory conditions in people from high- and middle-income countries. This correlation has led to studies that have identified proteins produced by hookworms that can suppress inflammatory bowel disease (IBD) and asthma in animal models. Hookworms secrete a family of abundant netrin-domain containing proteins referred to as AIPs (Anti-Inflammatory Proteins), but there is no information on the structure-function relationships. Here we have applied a downsizing approach to the hookworm AIPs to derive peptides of 20 residues or less, some of which display anti-inflammatory effects when co-cultured with human peripheral blood mononuclear cells and oral therapeutic activity in a chemically induced mouse model of acute colitis. Our results indicate that a conserved helical region is responsible, at least in part, for the anti-inflammatory effects. This helical region has potential in the design of improved leads for treating IBD and possibly other inflammatory conditions.

Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

Immune System Investigation Using Parasitic Helminths.

Coevolutionary adaptation between humans and helminths has developed a finely tuned balance between host immunity and chronic parasitism due to immunoregulation. Given that these reciprocal forces drive selection, experimental models of helminth infection are ideally suited for discovering how host protective immune responses adapt to the unique tissue niches inhabited by these large metazoan parasites. This review highlights the key discoveries in the immunology of helminth infection made over the last decade, from innate lymphoid cells to the emerging importance of neuroimmune connections. A particular emphasis is placed on the emerging areas within helminth immunology where the most growth is possible, including the advent of genetic manipulation of parasites to study immunology and the use of engineered T cells for therapeutic options. Lastly,we cover the status of human challenge trials with helminths as treatment for autoimmune disease, which taken together, stand to keep the study of parasitic worms at the forefront of immunology for years to come.

A netrin domain-containing protein secreted by the human hookworm Necator americanus protects against CD4 T cell transfer colitis.

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.

Treatment of mice with S4B6 IL-2 complex prevents lethal toxoplasmosis via IL-12- and IL-18-dependent interferon-gamma production by non-CD4 immune cells.

Toxoplasmic encephalitis is an AIDS-defining condition. The decline of IFN-γ-producing CD4+ T cells in AIDS is a major contributing factor in reactivation of quiescent Toxoplasma gondii to an actively replicating stage of infection. Hence, it is important to characterize CD4-independent mechanisms that constrain acute T. gondii infection. We investigated the in vivo regulation of IFN-γ production by CD8+ T cells, DN T cells and NK cells in response to acute T. gondii infection. Our data show that processing of IFN-γ by these non-CD4 cells is dependent on both IL-12 and IL-18 and the secretion of bioactive IL-18 in response to T. gondii requires the sensing of viable parasites by multiple redundant inflammasome sensors in multiple hematopoietic cell types. Importantly, our results show that expansion of CD8+ T cells, DN T cells and NK cell by S4B6 IL-2 complex pre-treatment increases survival rates of mice infected with T. gondii and this is dependent on IL-12, IL-18 and IFN-γ. Increased survival is accompanied by reduced pathology but is independent of expansion of TReg cells or parasite burden. This provides evidence for a protective role of IL2C-mediated expansion of non-CD4 cells and may represent a promising lead to adjunct therapy for acute toxoplasmosis.

Harnessing helminth-driven immunoregulation in the search for novel therapeutic modalities.

Parasitic helminths have coevolved with humans over millennia, intricately refining and developing an array of mechanisms to suppress or skew the host's immune system, thereby promoting their long-term survival. Some helminths, such as hookworms, cause little to no overt pathology when present in modest numbers and may even confer benefits to their human host. To exploit this evolutionary phenomenon, clinical trials of human helminth infection have been established and assessed for safety and efficacy for a range of immune dysfunction diseases and have yielded mixed outcomes. Studies of live helminth therapy in mice and larger animals have convincingly shown that helminths and their excretory/secretory products possess anti-inflammatory drug-like properties and represent an untapped pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins, glycans, post-translational modifications, and various metabolites. Although the concept of helminth-inspired therapies holds promise, it also presents a challenge to the drug development community, which is generally unfamiliar with foreign biologics that do not behave like antibodies. Identification and characterization of helminth molecules and vesicles and the molecular pathways they target in the host present a unique opportunity to develop tailored drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even so, much work remains to mine and assess this out-of-the-box therapeutic modality. Industry-based organizations need to consider long-haul investments aimed at unraveling and exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries with an eye on rapid and profitable turnarounds.

Randomized, Placebo Controlled Trial of Experimental Hookworm Infection for Improving Gluten Tolerance in Celiac Disease.

INTRODUCTION: Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled. METHODS: We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year). RESULTS: Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1-9); L3-20, 1 (0-9); and L3-40, 0 (0-3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, -0.6 [-1.3 to 0.2]; L3-20, -0.5 [-0.8 to 0.2]; and L3-40, -1.1 [-1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9-44] vs 45.9 [39.2-52.6]). DISCUSSION: Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses.

Inflammasome-Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung.

Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R-dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis, suggesting a potential role for the inflammasome in the IL-1-mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3 -/- mice with N. brasiliensis Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3 -/- mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3 -/- mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis, and in contrast to Nlrp3 -/- mice, antihelminth responses were unaffected in caspase-1/11-deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner.

Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1ß, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

The NLRP3 Inflammasome Suppresses Protective Immunity to Gastrointestinal Helminth Infection.

Inflammasomes promote immunity to microbial pathogens by regulating the function of IL-1-family cytokines such as IL-18 and IL-1ß. However, the roles for inflammasomes during parasitic helminth infections remain unclear. We demonstrate that mice and humans infected with gastrointestinal nematodes display increased IL-18 secretion, which in Trichuris-infected or worm antigen-treated mice and in macrophages co-cultured with Trichuris antigens or exosome-like vesicles was dependent on the NLRP3 inflammasome. NLRP3-deficient mice displayed reduced pro-inflammatory type 1 cytokine responses and augmented protective type 2 immunity, which was reversed by IL-18 administration. NLRP3-dependent suppression of immunity partially required CD4+ cells but was apparent even in Rag1-/- mice that lack adaptive immune cells, suggesting that NLRP3 influences both innate and adaptive immunity. These data highlight a role for NLRP3 in limiting protective immunity to helminths, suggesting that targeting the NLRP3 inflammasome may be an approach for limiting the disease burden associated with helminth infections.

Chronic Helminth Infection Perturbs the Gut-Brain Axis, Promotes Neuropathology, and Alters Behavior.

Helminth infections in children are associated with impaired cognitive development; however, the biological mechanisms for this remain unclear. Using a murine model of gastrointestinal helminth infection, we demonstrate that early-life exposure to helminths promotes local and systemic inflammatory responses and transient changes in the gastrointestinal microbiome. Behavioral and cognitive analyses performed 9-months postinfection revealed deficits in spatial recognition memory and an anxiety-like behavioral phenotype in worm-infected mice, which was associated with neuropathology and increased microglial activation within the brain. This study demonstrates a previously unrecognized mechanism through which helminth infections may influence cognitive function, via perturbations in the gut-immune-brain axis.

Polypyridylruthenium(II) complexes exert in vitro and in vivo nematocidal activity and show significant inhibition of parasite acetylcholinesterases.

Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb12-mono, which was among the most potent in its ability to kill L3 (IC50 = 3.1 ± 0.4 µM) and adult (IC50 = 5.2 ± 0.3 µM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb12-mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significant decreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance.

Helminth Immunomodulation in Autoimmune Disease.

Helminths have evolved to become experts at subverting immune surveillance. Through potent and persistent immune tempering, helminths can remain undetected in human tissues for decades. Redirecting the immunomodulating "talents" of helminths to treat inflammatory human diseases is receiving intensive interest. Here, we review therapies using live parasitic worms, worm secretions, and worm-derived synthetic molecules to treat autoimmune disease. We review helminth therapy in both mouse models and clinical trials and discuss what is known on mechanisms of action. We also highlight current progress in characterizing promising new immunomodulatory molecules found in excretory/secretory products of helminths and their potential use as immunotherapies for acute and chronic inflammatory diseases.

Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma.

In the developed world, declining prevalence of some parasitic infections correlates with increased incidence of allergic and autoimmune disorders. Moreover, experimental human infection with some parasitic worms confers protection against inflammatory diseases in phase 2 clinical trials. Parasitic worms manipulate the immune system by secreting immunoregulatory molecules that offer promise as a novel therapeutic modality for inflammatory diseases. We identify a protein secreted by hookworms, anti-inflammatory protein-2 (AIP-2), that suppressed airway inflammation in a mouse model of asthma, reduced expression of costimulatory markers on human dendritic cells (DCs), and suppressed proliferation ex vivo of T cells from human subjects with house dust mite allergy. In mice, AIP-2 was primarily captured by mesenteric CD103+ DCs and suppression of airway inflammation was dependent on both DCs and Foxp3+ regulatory T cells (Tregs) that originated in the mesenteric lymph nodes (MLNs) and accumulated in distant mucosal sites. Transplantation of MLNs from AIP-2-treated mice into naïve hosts revealed a lymphoid tissue conditioning that promoted Treg induction and long-term maintenance. Our findings indicate that recombinant AIP-2 could serve as a novel curative therapeutic for allergic asthma and potentially other inflammatory diseases.

Identification of lead chemotherapeutic agents from medicinal plants against blood flukes and whipworms.

Schistosomiasis and trichuriasis are two of the most common neglected tropical diseases (NTD) that affect almost a billion people worldwide. There is only a limited number of effective drugs to combat these NTD. Medicinal plants are a viable source of parasiticides. In this study, we have investigated six of the 19 phytochemicals isolated from two Bhutanese medicinal plants, Corydalis crispa and Pleurospermum amabile, for their anthelmintic properties. We used the xWORM technique and Scanning Electron Microscope-based imaging to determine the activity of the compounds. Of the six compounds tested, isomyristicin and bergapten showed significant anthelmintic activity against Schistosoma mansoni and Trichuris muris with bergapten being the most efficacious compound one against both parasites (S. mansoni IC50 = 8.6 µg/mL and T. muris IC50 = 10.6 µg/mL) and also against the schistosomulum stage of S. mansoni. These two compounds induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. The efficacy against multiple phylogenetically distinct parasites and different life stages, especially the schistosomulum where praziquantel is ineffective, makes isomyristicin and bergapten novel scaffolds for broad-spectrum anthelmintic drug development that could be used for the control of helminths infecting humans and animals.

Compounds Derived from the Bhutanese Daisy, Ajania nubigena, Demonstrate Dual Anthelmintic Activity against Schistosoma mansoni and Trichuris muris.

BACKGROUND: Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds-luteolin (3) and (3R,6R)-linalool oxide acetate (1)-showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8-36.9 µg/mL) and T. muris (IC50 range = 9.7-20.4 µg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 µg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice. CONCLUSIONS/SIGNIFICANCE: Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths-T. muris and S. mansoni-and was effective against juvenile schistosomes, the stage that is refractory to the current gold standard drug, praziquantel. Medicinal chemistry optimisation including cytotoxicity analysis, analogue development and structure-activity relationship studies are warranted and could lead to the identification of more potent chemical entities for the control of parasitic helminths of humans and animals.