Exploring the antifilarial potential of an important medicinal plant Typhonium trilobatum (L. Schoot): Isolation, characterization, and structural elucidation of bioactive compounds against Brugia malayi.

ETHNOPHARMACOLOGY RELEVANCE: The plant Typhonium trilobatum has been utilized in traditional medicine for the treatment of many ailments, including parasitic infections. Recent examinations indicate that the bioactive substances from this plant may have antiparasitic activities against Brugia malayi, which have not been determined. PURPOSE: The parasitic nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti causing lymphatic filariasis, remain a significant challenge to global public health. Given the ongoing nature of this enduring menace, the current research endeavours to examine the efficacy of an important medicinal plant, Typhonium trilobatum. METHODS: Different extracts of the T. trilobatum tubers were evaluated for their antiparasitic activity. The most prominent extract was subjected to Gas Chromatography Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC) followed by Column Chromatography for isolating bioactive molecules. The major compounds were isolated and characterized based on different spectroscopic techniques (FTIR, NMR and HRMS). Further, the antiparasitic activity of the isolated compounds was evaluated against B. malayi and compared with clinically used antifilarial drugs like Diethylcarbamazine and Ivermectin. RESULTS: The methanolic extract of the tuber exhibited significant antiparasitic activity compared to the other extracts. The bioactive molecules isolated from the crude extract were identified as Linoleic acid and Palmitic acid. Antiparasitic activity of both the compounds has been performed against B. malayi and compared with clinically used antifilarial drugs, Ivermectin and DEC. The IC50 value of Linoleic acid was found to be 6.09 ± 0.78 µg/ml after 24 h and 4.27 ± 0.63 µg/ml after 48 h, whereas for Palmitic acid the value was 12.35 ± 1.09 µg/ml after 24 h and 8.79 ± 0.94 µg/ml after 48 h. The IC50 values of both the molecules were found to be similar to the standard drug Ivermectin (IC50 value of 11.88 ± 1.07 µg/ml in 24 h and 2.74 ± 0.43 µg/ml in 48 h), and much better compared to the DEC (IC50 values of 194.2 ± 2.28 µg/ml in 24 h and 101.8 ± 2.06 µg/ml in 48 h). Furthermore, it has been observed that both the crude extracts and the isolated compounds do not exhibit any detrimental effects on the J774.A.1 macrophage cell line. CONCLUSION: The isolation and characterization of bioactive compounds present in the methanolic tuber extract of Typhonium trilobatum were explored. Moreover, the antimicrofilarial activity of the crude extracts and its two major compounds were determined using Brugia malayi microfilarial parasites without any significant side effects.

Biophysical and in-silico studies on the structure-function relationship of Brugia malayi protein disulfide isomerase.

Human Lymphatic filariasis is caused by parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Protein disulfide isomerase (PDI), a redox-active enzyme, helps to form and isomerize the disulfide bonds, thereby acting as a chaperone. Such activity is essential for activating many essential enzymes and functional proteins. Brugia malayi protein disulfide isomerase (BmPDI) is crucial for parasite survival and an important drug target. Here, we used a combination of spectroscopic and computational analysis to study the structural and functional changes in the BmPDI during unfolding. Tryptophan fluorescence data revealed two well-separated transitions during the unfolding process, suggesting that the unfolding of the BmPDI is non-cooperative. The binding of the fluorescence probe 8-anilino-1-naphthalene sulfonic acid dye (ANS) validated the results obtained by the pH unfolding. The dynamics of molecular simulation performed at different pH conditions revealed the structural basis of BmPDI unfolding. Detailed analysis suggested that under different pH, both the global structure and the conformational dynamics of the active site residues were differentially altered. Our multiparametric study reveals the differential dynamics and collective motions of BmPDI unfolding, providing insights into its structure-function relationship.Communicated by Ramaswamy H. Sarma.

Surveillance and Selective Treatment of Brugia malayi Filariasis Eleven Years after Stopping Mass Drug Administration in Belitung District, Indonesia.

Brugia malayi is the major cause of lymphatic filariasis (LF) in Indonesia. Zoophilic B. malayi was endemic in Belitung district, and mass drug administration (MDA) with diethylcarbamazine (DEC) and albendazole ceased after five annual rounds in 2010. The district passed three transmission assessment surveys (TAS) between 2011 and 2016. As part of the post-TAS3 surveillance of the national LF elimination program, we collected night blood samples for microfilaria (Mf) detection from 1,911 subjects more than 5 years of age in seven villages. A B. malayi Mf prevalence ranging from 1.7% to 5.9% was detected in five villages. Only 2 (5%) of the total 40 Mf-positive subjects were adolescents aged 18 and 19 years old, and 38 (95%) Mf-positive subjects were 21 years and older. Microfilarial densities in infected individuals were mostly low, with 60% of the subjects having Mf densities between 16 and 160 Mf/mL. Triple-drug treatment with ivermectin, DEC, and albendazole (IDA) was given to 36 eligible Mf-positive subjects. Adverse events were mostly mild, and treatment was well tolerated. One year later, 35 of the treated Mf-positive subjects were reexamined, and 33 (94%) had cleared all Mf, while the anti-Bm14 antibody prevalence remained almost unchanged. Results indicate that in B. malayi-endemic areas, post-TAS3 surveillance for Mf in the community may be needed to detect a potential parasite reservoir in adults. Selective treatment with IDA is highly effective in clearing B. malayi Mf and should be used to increase the prospects for LF elimination if MDA is reintroduced.

Prevalence of zoonotic (brugian) filariasis in Asia: A proportional meta-analysis.

Lymphatic filariasis is a public health problem and targeted for global elimination. WHO recommends mass drug administration to interrupt transmission of the parasites involved. There are concerns that transmission interruption may be difficult in areas of zoonotic filarial infections. This study aimed to estimate the pooled prevalence of zoonotic brugian filariasis, and to compare the pooled prevalence of brugian filariasis in human and animal populations in the same area based on available studies. A comprehensive literature search was conducted in health-related electronic databases (PubMed, Ovid MEDLINE, Index Medicus, google scholar). A random-effect meta-analysis of the pooled overall prevalence of filariasis in animal populations was conducted. Sixteen studies from four different Asian countries were identified. Studies were conducted most frequently in Thailand (n = 7), followed by Malaysia (n = 5), India (n = 3), and Sri Lanka (n = 1). Regardless of animal group, the pooled overall prevalence of animal Brugia infections was 13% (95%CI: 7-21%, I2:98%, 16 studies). On stratification, the pooled overall prevalence in the animal population was 19% (95%CI: 1-50%, I2: 99%, 3 studies) in India, 8% (95%CI: 2-7%, I2: 97%, 5 studies) in Malaysia, and 13% (95%CI: 7-20%, I2: 94%, 7 studies) in Thailand. The prevalence in the animal population was 17% (95%CI: 13-21%, 1 study) in Sri Lanka. The pooled overall prevalence of Brugia malayi was 13% (95%CI: 7-21%, I2:98%, 12 studies), while for Brugia pahangi this was 12% (95%CI: 7-19%, I2:86%, 7 studies). Regardless of animal group, geographic area, or diagnostic test, the prevalence of B. malayi was consistently high. On stratification by animal category, the pooled overall prevalence was 10% (95%CI: 6-14%, I2:92%, 13 studies) in cats, 12% (95%CI: 2-28%, I2: 99%, 6 studies) in dogs, and 55% (95%CI: 47-63%, 1 study) in leaf-eating monkeys. The findings show the extent of zoonotic Brugiainfections in domestic cats and dogs, suggesting that these animals are potential reservoirs for human brugian filariasis in the study countries. To substantiate this with more accuracy, future well designed whole genomic sequencing of individual mf collected from humans and B. malayi infected animals in the same area are needed.

Immune Response Profiling of Cocktails of Brugia malayi Vaccine Candidates DIM-1, Calponin and Troponin 1 in BALB/c Mice.

PURPOSE: In search of a vaccine for the control of human lymphatic filariasis (LF) caused by Wuchereria bancrofti, Brugia malayi and B. timori, we identified three parasite-specific potential candidates: the disorganized muscle protein-1 (D), calponin (C) and troponin 1 (T) in B. malayi adult worm. In the present study, we investigated the immune response profile of the cocktails of the recombinant D, T and C proteins. METHODS: Groups of BALB/c mice were immunized with individual rproteins or their cocktails DT, TC, DC and DTC, and the immunogen-specific IgG and its subclasses and IgE were determined. Cells from the immunized animals were challenged in vitro with the respective rproteins and cocktails and the release of nitric oxide (NO) from macrophages and Th1 and Th2 cytokines from splenocytes were determined. RESULTS: Among the immunized groups, DTC elicited comparatively a stronger response which included augmented release of NO, Th1 (IL-1ß, IL-2, IFN-γ and TNF-α) and Th2 (IL-4, IL-6, IL-10 and TGF-ß) cytokines, and increased levels of immunogen-specific IgG, IgG1 and IgG2b and low levels of immunogen-specific IgG2a and IgE and the Th2 cytokine IL-13. CONCLUSION: Immune responses that play important role in host protection were elicited strongly by DTC cocktail compared to the individual rproteins or DT, TC and DC cocktails. The findings provide a sound rationale for further studies on DTC cocktail as a vaccine candidate for the control of LF.

Diethylcarbamazine elicits Ca2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles.

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals' lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.

Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.

Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite Brugia malayi to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.

Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi.

N-linked glycosylation is a critical post translational modification of eukaryotic proteins. N-linked glycans are present on surface and secreted filarial proteins that play a role in host parasite interactions. Examples of glycosylated Brugia malayi proteins have been previously identified but there has not been a systematic study of the N-linked glycoproteome of this or any other filarial parasite. In this study, we applied an enhanced N-glyco FASP protocol using an engineered carbohydrate-binding protein, Fbs1, to enrich N-glycosylated peptides for analysis by LC-MS/MS. We then mapped the N-glycosites on proteins from three host stages of the parasite: adult female, adult male and microfilariae. Fbs1 enrichment of N-glycosylated peptides enhanced the identification of N-glycosites. Our data identified 582 N-linked glycoproteins with 1273 N-glycosites. Gene ontology and cell localization prediction of the identified N-glycoproteins indicated that they were mostly membrane and extracellular proteins. Comparing results from adult female worms, adult male worms, and microfilariae, we find variability in N-glycosylation at the protein level as well as at the individual N-glycosite level. These variations are highlighted in cuticle N-glycoproteins and adult worm restricted N-glycoproteins as examples of proteins at the host parasite interface that are well positioned as potential therapeutic targets or biomarkers.

Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host.

Vector-borne, filarial nematode diseases cause significant disease burdens in humans and domestic animals worldwide. Although there is strong direct evidence of parasite-driven immunomodulation of mammalian host responses, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a filarial nematode and causative agent of Lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae-derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an Ae. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs. AAEL002590, an Ae. aegypti gene encoding a serine protease, was shown to be downregulated when cells were treated with biologically relevant EV concentrations in vitro. Injection of adult female mosquitoes with biologically relevant concentrations of EVs validated these results in vivo, recapitulating the downregulation of AAEL002590 transcript. This gene was predicted to be involved in the mosquito phenoloxidase (PO) cascade leading to the canonical melanization response and correspondingly, both suppression of this gene using RNAi and parasite EV treatment reduced PO activity in vivo. Our data indicate that parasite-derived EVs interfere with critical immune responses in the vector host, including melanization.

Wolbachia Ferrochelatase as a potential drug target against filarial infections.

Filarial infections are among the world's most disturbing diseases caused by 3 major parasitic worms; Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi, affecting more than 500 million people worldwide. Currently used drugs for mass drug administration (MDA) have been met with several challenges including the development of complications in individuals with filaria co-infections and parasitic drug resistance. The filarial endosymbiont, Wolbachia, has emerged as an attractive therapeutic target for filariasis elimination, due to the dependence of the filaria on this endosymbiont for survival. Here, we target an important enzyme in the Wolbachia heme biosynthetic pathway (ferrochelatase), using high-throughput virtual screening and molecular dynamics with MM-PBSA calculations. We identified four drug candidates; Nilotinib, Ledipasvir, 3-benzhydryloxy-8-methyl-8-azabicyclo[3.2.1]octane, and 2-(4-Amino-piperidin-1-yl)-ethanol as potential small molecules inhibitors as they could compete with the enzyme's natural substrate (Protoporphyrin IX) for active pocket binding. This prevents the worm from receiving the heme molecule from Wolbachia for their growth and survival, resulting in their death. This study which involved targeting enzymes in biosynthetic pathways of the parasitic worms' endosymbiont (Wolbachia), has proven to be an alternative therapeutic option leading to the discovery of new drugs, which will help facilitate the elimination of parasitic infections.

Human immune response against filarial HSP70 and its role in the diagnosis of lymphatic filariasis.

A sensitive and specific diagnostic kit is crucial for the detection of human lymphatic filariasis at the early stage of infection as the existing diagnostic tools are inefficient and expensive. In the present study, we have cloned and expressed Brugia malayi HSP70 (BmHSP70) protein and characterized it as a potential antigen for diagnosis of the asymptomatic microfilariae stage of Wuchereria. bancrofti infection using ELISA, western blot, and bioinformatics tools. The antigenic efficacy of BmHSP70 was also compared with ScHSP70. The BmHSP70 and ScHSP70 peptide showed highly antigenic in nature and they showed immunogenic cross-reactivity endemic normal (EN) < chronic (CH) < microfilaraemic (MF) in IgG, IgG1, and IgG4 ELISA. IgG4-specific immunoblotting of BmHSP70 with MF sera further explicated its stage-specific antigenic cross-reactivity. These antigens (ScHSP70 and BmHSP70) showed a positive immunogenic correlation with the number of MF in blood samples. Thus, proposing BmHSP70 as a potential immunodiagnostic antigen against lymphatic filariasis. A triplet of GGMP tetrapeptide specific to the filarial HSP70 was also identified which was absent in human HSP70. In terms of sensitivity and specificity of antigens, these results suggest that recombinant BmHSP70 is a good antigen and could be used to diagnose early-stage of microfilariae infection.

Unraveling cross-reactivity of anti-glycan IgG responses in filarial nematode infections.

Parasitic nematodes responsible for filarial diseases cause chronic disablement in humans worldwide. Elimination programs have substantially reduced the rate of infection in certain areas, but limitations of current diagnostics for population surveillance have been pointed out and improved assays are needed to reach the elimination targets. While serological tests detecting antibodies to parasite antigens are convenient tools, those currently available are compromised by the occurrence of antibodies cross-reactive between nematodes, as well as by the presence of residual antibodies in sera years after treatment and clearance of the infection. We recently characterized the N-linked and glycosphingolipid derived glycans of the parasitic nematode Brugia malayi and revealed the presence of various antigenic structures that triggered immunoglobulin G (IgG) responses in infected individuals. To address the specificity of IgG binding to these glycan antigens, we screened microarrays containing Brugia malayi glycans with plasma from uninfected individuals and from individuals infected with Loa loa, Onchocerca volvulus, Mansonella perstans and Wuchereria bancrofti, four closely related filarial nematodes. IgG to a restricted subset of cross-reactive glycans was observed in infection plasmas from all four species. In plasma from Onchocerca volvulus and Mansonella perstans infected individuals, IgG binding to many more glycans was additionally detected, resulting in total IgG responses similar to the ones of Brugia malayi infected individuals. For these infection groups, Brugia malayi, Onchocerca volvulus and Mansonella perstans, we further studied the different IgG subclasses to Brugia malayi glycans. In all three infections, IgG1 and IgG2 appeared to be the major subclasses involved in response to glycan antigens. Interestingly, in Brugia malayi infected individuals, we observed a marked reduction in particular in IgG2 to parasite glycans post-treatment with anthelminthic, suggesting a promising potential for diagnostic applications. Thus, we compared the IgG response to a broad repertoire of Brugia malayi glycans in individuals infected with various filarial nematodes. We identified broadly cross-reactive and more specific glycan targets, extending the currently scarce knowledge of filarial nematode glycosylation and host anti-glycan antibody response. We believe that our initial findings could be further exploited to develop disease-specific diagnostics as part of an integrated approach for filarial disease control.

Wbm0076, a candidate effector protein of the Wolbachia endosymbiont of Brugia malayi, disrupts eukaryotic actin dynamics.

Brugia malayi, a parasitic roundworm of humans, is colonized by the obligate intracellular bacterium, Wolbachia pipientis. The symbiosis between this nematode and bacterium is essential for nematode reproduction and long-term survival in a human host. Therefore, identifying molecular mechanisms required by Wolbachia to persist in and colonize B. malayi tissues will provide new essential information regarding the basic biology of this endosymbiosis. Wolbachia utilize a Type IV secretion system to translocate so-called "effector" proteins into the cytosol of B. malayi cells to promote colonization of the eukaryotic host. However, the characterization of these Wolbachia secreted proteins has remained elusive due to the genetic intractability of both organisms. Strikingly, expression of the candidate Wolbachia Type IV-secreted effector protein, Wbm0076, in the surrogate eukaryotic cell model, Saccharomyces cerevisiae, resulted in the disruption of the yeast actin cytoskeleton and inhibition of endocytosis. Genetic analyses show that Wbm0076 is a member of the family of Wiskott-Aldrich syndrome proteins (WAS [p]), a well-conserved eukaryotic protein family required for the organization of actin skeletal structures. Thus, Wbm0076 likely plays a central role in the active cell-to-cell movement of Wolbachia throughout B. malayi tissues during nematode development. As most Wolbachia isolates sequenced to date encode at least partial orthologs of wBm0076, we find it likely that the ability of Wolbachia to directly manipulate host actin dynamics is an essential requirement of all Wolbachia endosymbioses, independent of host cell species.

Pharmacological Profiling of a Brugia malayi Muscarinic Acetylcholine Receptor as a Putative Antiparasitic Target.

The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor of B. malayi (Bma-GAR-3) that is highly expressed across intramammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Finally, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

Diethylcarbamazine, TRP channels and Ca2+ signaling in cells of the Ascaris intestine.

The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine.

Diethylcarbamazine mediated potentiation of emodepside induced paralysis requires TRP-2 in adult Brugia malayi.

Human and veterinary filarial nematode infections are a major health concern in tropical countries. They are transmitted by biting insects and mosquitoes. Lymphatic filariasis, a group of filarial infections caused by Brugia spp. and Wucheria bancrofti affect more than 120 million people worldwide. Infected individuals develop swollen limbs and disfigurement, leading to an inability to work and ostracization from society. Control and prophylaxis for these infections involve mass drug administration combinations of anthelmintics including diethylcarbamazine (DEC). DEC has actions on microfilariae, but its effects on adult worms are less pronounced. The SLO-1 (BK) channel activator, emodepside, kills adults of many filarial species. However, the in vivo efficacy of emodepside is suboptimal against B. malayi, possibly due to reduced bioavailability in the lymphatic system. Expressing different slo-1 splice variants in B. malayi also affects sensitivity to emodepside. This study explores the potentiation of emodepside mediated paralysis by DEC in adult female B. malayi. Worminator motility measurements show that co-application of DEC and emodepside increases the potency of emodepside 4-fold. The potentiation of the emodepside effect persists even after the worms recover (desensitize) from the initial effects of DEC. RNAi knock-down demonstrates that the DEC-mediated potentiation of emodepside requires the presence of TRP-2 channels. Our study demonstrates that the addition of DEC could enhance the effect of emodepside where bioavailability or activity against a specific species may be low.

Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites.

Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal response to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to reach larger response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that an issue with synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to new subunit combinations that lead to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

Immunomodulatory effects of chitosan nanoparticles as vaccine delivery agent against lymphatic filariasis through mucosal immunization.

Lymphatic filariasis is a parasitic disease caused by nematodes affecting millions of individuals in the tropical region. The complex life cycle of the filarial parasite eludes protective measures such as chemotherapy and vector control. Vaccination through recombinant proteins stands as one of the safe and most effective methods. The filarial antigens Brugia malayi Thioredoxin (TRX) and abundant larval transcript-2 (ALT-2) can induce recognizable levels of protection in murine animal models. Chitosan is a safe, non-toxic material ubiquitously served as an efficient carrier and an adjuvant. The present study was attempted to enhance the immune efficacy of filarial antigens using chitosan nanoparticles (CN) through mucosal routes of immunization. Our study showed that oral immunization was able to produce enhanced humoral response and balanced Th1/Th2 antibody isotype response for the recombinant antigens compared to intranasal routes. A high level of splenocyte T cell proliferation (P < 0.01) was obtained for both routes. The cytokine analysis showed a high level of IFN-γ followed by IL-5 for the oral route, whereas a high level of IL-4 was observed for intranasal route. These results confirm the ability of chitosan nanoparticles to elevate the immune efficacy of the antigens through the oral route in mice.

Alteration of rhesus macaque serum N-glycome during infection with the human parasitic filarial nematode Brugia malayi.

Serum N-glycan profiling studies during the past decades have shown robust associations between N-glycan changes and various biological conditions, including infections, in humans. Similar studies are scarcer for other mammals, despite the tremendous potential of serum N-glycans as biomarkers for infectious diseases in animal models of human disease and in the veterinary context. To expand the knowledge of serum N-glycan profiles in important mammalian model systems, in this study, we combined MALDI-TOF-MS analysis and HILIC-UPLC profiling of released N-glycans together with glycosidase treatments to characterize the glycan structures present in rhesus macaque serum. We used this baseline to monitor changes in serum N-glycans during infection with Brugia malayi, a parasitic nematode of humans responsible for lymphatic filariasis, in a longitudinal cohort of infected rhesus macaques. Alterations of the HILIC-UPLC profile, notably of abundant structures, became evident as early as 5 weeks post-infection. Given its prominent role in the immune response, contribution of immunoglobulin G to serum N-glycans was investigated. Finally, comparison with similar N-glycan profiling performed during infection with the dog heartworm Dirofilaria immitis suggests that many changes observed in rhesus macaque serum N-glycans are specific for lymphatic filariasis.

Nodulisporic acid produces direct activation and positive allosteric modulation of AVR-14B, a glutamate-gated chloride channel from adult Brugia malayi.

Glutamate-gated chloride channels (GluCls) are unique to invertebrates and are targeted by macrocyclic lactones. In this study, we cloned an AVR-14B GluCl subunit from adult Brugia malayi, a causative agent of lymphatic filariasis in humans. To elucidate this channel's pharmacological properties, we used Xenopus laevis oocytes for expression and performed two-electrode voltage-clamp electrophysiology. The receptor was gated by the natural ligand L-glutamate (effective concentration, 50% [EC50] = 0.4 mM) and ivermectin (IVM; EC50 = 1.8 nM). We also characterized the effects of nodulisporic acid (NA) on Bma-AVR-14B and NA-produced dual effects on the receptor as an agonist and a type II positive allosteric modulator. Here we report characterization of the complex activity of NA on a nematode GluCl. Bma-AVR-14B demonstrated some unique pharmacological characteristics. IVM did not produce potentiation of L-glutamate-mediated responses but instead, reduced the channel's sensitivity for the ligand. Further electrophysiological exploration showed that IVM (at a moderate concentration of 0.1 nM) functioned as an inhibitor of both agonist and positive allosteric modulatory effects of NA. This suggests that IVM and NA share a complex interaction. The pharmacological properties of Bma-AVR-14B indicate that the channel is an important target of IVM and NA. In addition, the unique electrophysiological characteristics of Bma-AVR-14B could explain the observed variation in drug sensitivities of various nematode parasites. We have also shown the inhibitory effects of IVM and NA on adult worm motility using Worminator. RNA interference (RNAi) knockdown suggests that AVR-14 plays a role in influencing locomotion in B. malayi.