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.

Stigma, depression and quality of life among people affected by neglected tropical diseases in Nepal.

BACKGROUND: Stigma and poor mental health are important factors influencing the quality of life (QOL) of people with neglected tropical diseases (NTDs). This study examines the relationship between stigma, depression and QOL among people affected by leprosy and lymphatic filariasis (LF) in Nepal. METHODS: A cross-sectional community-based survey was conducted among 102 NTD-affected persons (70 leprosy and 32 LF) using interview-administered questionnaires measuring the level of stigma (5-QSI-AP), symptoms of depression (PHQ-9) and QOL (WHOQOL-8). Three different regression models were developed, each using the ordinary least squares and Poisson regression to evaluate the association between stigma and QOL, depression and QOL, and stigma and depression. RESULTS: The mean scores were 21.8±4.4 for QOL, 6.6±5.6 for depression and 3.0±2.8 for stigma. Almost 17% reported the prevalence of depression symptoms. Both stigma (ß=-0.65, p<0.001) and depression (ß=-0.32, p<0.001) were associated with lower scores for QOL, while there was a significant positive relationship between stigma and depression (ß=0.92, p<0.001). Similar results were obtained from the Poisson regression models. CONCLUSIONS: The study showed a considerable burden of depression, stigma and poor QOL among study participants with leprosy and LF in Nepal. A holistic package of care that addresses the physical, mental and psychological well-being of people with NTD is required. CONTEXTE: La stigmatisation et la mauvaise santé mentale sont des facteurs importants qui influencent la qualité de vie des personnes atteintes de maladies tropicales négligées (MTN). Cette étude examine la relation entre la stigmatisation, la dépression et la qualité de vie chez les personnes atteintes de lèpre et de filariose lymphatique au Népal. MÉTHODES UTILISÉES: Une enquête communautaire transversale a été menée auprès de 102 personnes atteintes de MTN (70 de la lèpre et 32 de la filariose lymphatique) à l'aide de questionnaires administrés par entretien mesurant le niveau de stigmatisation (5-QSI-AP), les symptômes de dépression (PHQ-9) et la qualité de vie (WHOQOL-8). Trois modèles de régression différents ont été développés, chacun utilisant les moindres carrés ordinaires et la régression de Poisson pour évaluer l'association entre : (i) la stigmatisation et la QV; (ii) la dépression et la QV; et (iii) la stigmatisation et la dépression. RÉSULTATS: Les scores moyens étaient de 21,8±4,4 pour la QV, 6,6±5,6 pour la dépression, et 3,0±2,8 pour la stigmatisation. Près de 17% des personnes interrogées ont signalé la prévalence de symptômes dépressifs. La stigmatisation (ß = -0,65, p<0 001) et la dépression (ß = -0,32, p<0 001) ont été associées à des scores plus faibles pour la qualité de vie, tandis qu'il existait une relation positive significative entre la stigmatisation et la dépression (ß = 0,92, p<0 001). Des résultats similaires ont été obtenus à partir des modèles de régression de Poisson. CONCLUSIONS: L'étude a montré une incidence importante de dépression, de stigmatisation et d'une mauvaise qualité de vie parmi les participants atteints de lèpre et de FL au Népal. Il convient donc de mettre en place un ensemble de soins holistiques pour ces patients qui prendrait en compte le bien-être physique, mental et psychologique des personnes atteintes de MTN. ANTECEDENTES: La estigmatización y la mala salud mental son factores importantes que influyen en la calidad de vida de las personas con enfermedades tropicales desatendidas. las personas con enfermedades tropicales desatendidas (ETD). Este estudio examina la relación entre el estigma, la depresión y la CdV entre las personas afectadas por lepra y lepra y la filariasis linfática en Nepal. MÉTODOS: Se realizó una encuesta comunitaria transversal entre 102 personas afectadas por ETD (70 de lepra y 32 de filariasis linfática) utilizando cuestionarios entrevistas para medir el nivel de estigma (5-QSI-AP), los síntomas de depresión (PHQ- 9) y la CdV (OMS- 9). 9) y la calidad de vida (WHOQOL-8). Se desarrollaron tres modelos de regresión diferentes regresión de Poisson para evaluar la asociación entre: (i) el estigma y (ii) la depresión. entre: (i) estigma y CdV; (ii) depresión y CdV; y (iii) estigma y depresión. RESULTADOS: Las puntuaciones medias fueron 21,8±4,4 para la CdV, 6,6±5,6 para la depresión y 3,0±2,8 para el estigma. Casi el 17% informó de la prevalencia de síntomas de depresión. Tanto el estigma (ß = -0,65, p<0 001), como la depresión (ß = -0,32, p<0 001) se asociaron con puntuaciones más bajas para la CdV, mientras que hubo una relación positiva significativa entre el estigma y la depresión (ß = 0,92, p<0 001). Se obtuvieron resultados similares en los modelos de regresión de Poisson. CONCLUSIONES: El estudio mostró una carga considerable de depresión, estigma y mala CdV entre los participantes del estudio con lepra y FL en Nepal. Se requiere un paquete holístico de atención que aborde el bienestar físico, mental y psicológico de las personas con ETD.

Larvicidal Activities of Allium sativum L. and Zingiber officinale Rosc. Extracts against Filariasis Vectors in Hadiya Zone, Ethiopia.

Mosquitoes present an immense threat to millions of people worldwide and act as vectors for filariasis disease. The objective of the study was to determine the effect of Allium sativum and Zingiber officinale extracts against filariasis vectors. The larvae were collected from the breeding site by using standard procedures for identification and larvicidal activities. Twenty grams (20 g) from each (Allium sativum and Zingiber officinale) were extracted separately by aqueous, ethanol, and methanol solvents. The phytochemical analysis was determined in the crude sample by using standard methods. Then, larvicidal effects were determined by introducing 10 larvae of the vectors to the concentrations of 250 ppm, 500 ppm, and 750 ppm of the crude sample, and data were subjected to probit analysis to determine the LC50 and Chi-squared test to check the significance of the mortality by R software. Anopheles funestus, Anopheles gambiae s.l., Anopheles pharoensis, Culex antennatus, and Culex quinquefasciatus were the filariasis vectors identified during the study period. The presence of phytochemical tests such as anthraquinones, flavonoids, glycosides, phenol, saponin, steroids, tannin, and terpenes was obtained. The larvicidal effects of the selected plant extracts ranged from 0%-100%. The lowest LC50 (53 ppm) was observed for A. sativum methanol test extract against Cx. quinquefasciatus. Ethanol extracts of A. sativum have a significant effect on An. funestus (X2 = 7.5, p = 0.02352) and Cx. quinquefasciatus (X2 = 10.833, p = 0.0.0044), whereas aqueous extracts have a significant effect only on An. gambiae s.l. (X2 = 7.0807, p = 0.029. Ethanol extracts of Z. officinale have a significant effect only on the mortality of An. pharoensis (X2 = 7.0807, p = 0.029), but methanol and aqueous extracts have no significant effect against filariasis vectors. In conclusion, A. sativum have a high toxic effect than Z. officinale extract against filariasis vectors in all type of solvents. So using those plant extracts is the best to reduce the risk of the synthetic chemical on nontarget organisms and the environment, in addition to the control of mosquito-borne diseases, but further studies will be conducted to evaluate the toxicity at different stages of the vectors.

Bio-efficacy of insecticidal molecule emodin against dengue, filariasis, and malaria vectors.

Emodin, a compound isolated from Aspergillus terreus, was studied using chromatographic and spectroscopic methods and compound purity (96%) was assessed by TLC. Furthermore, high larvicidal activity against Aedes aegypti-AeA (LC50 6.156 and LC90 12.450 mg/L), Culex quinquefasciatus-CuQ (8.216 and 14.816 mg/L), and Anopheles stephensi-AnS larvae (6.895 and 15.24 mg/L) was recorded. The first isolated fraction (emodin) showed higher pupicidal activity against AeA (15.449 and 20.752 mg/L). Most emodin-treated larvae (ETL) showed variations in acetylcholine esterase, α and ß-carboxylesterases, and phosphatase activities in the 4th instar, indicating the intrinsic differences in their biochemical changes. ETL had numerous altered tissues, including muscle, gastric caeca, hindgut, midgut, nerve ganglia, and midgut epithelium. Acute toxicity of emodin on brine shrimp Artemia nauplii (54.0 and 84.5 mg/L) and the zebrafish Danio rerio (less toxicity observed) was recorded. In docking studies, Emodin interacted well with odorant-binding-proteins of AeA, AnS, and CuQ with docking scores of - 8.89, - 6.53, and - 8.09 kcal mol-1, respectively. Therefore, A. terreus is likely to be effective against mosquito larvicides.

Modeling the metabolic interplay between a parasitic worm and its bacterial endosymbiont allows the identification of novel drug targets.

The filarial nematode Brugia malayi represents a leading cause of disability in the developing world, causing lymphatic filariasis in nearly 40 million people. Currently available drugs are not well-suited to mass drug administration efforts, so new treatments are urgently required. One potential vulnerability is the endosymbiotic bacteria Wolbachia-present in many filariae-which is vital to the worm. Genome scale metabolic networks have been used to study prokaryotes and protists and have proven valuable in identifying therapeutic targets, but have only been applied to multicellular eukaryotic organisms more recently. Here, we present iDC625, the first compartmentalized metabolic model of a parasitic worm. We used this model to show how metabolic pathway usage allows the worm to adapt to different environments, and predict a set of 102 reactions essential to the survival of B. malayi. We validated three of those reactions with drug tests and demonstrated novel antifilarial properties for all three compounds.

In vivo efficacy of the boron-pleuromutilin AN11251 against Wolbachia of the rodent filarial nematode Litomosoides sigmodontis.

The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal-adult worm killing-drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10-14 days. Therefore, AN11251 represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis.

Effect of tiliamosine, a bis, benzylisoquinoline alkaloid isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom on the immature stages of filarial mosquito Culex quinquefasciatus say (Diptera: Culicidae).

The present study was aimed to check the mosquitocidal activity of tiliamosine isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom against immature stages of Culex quinquefasciatus. Eggs and larvae of Cx. quinquefasciatus were exposed to different concentrations of tiliamosine - 0.5, 1.0, 1.5 and 2.0 ppm - prepared using DMSO. The compound tiliamosine showed good larvicidal activity with LC50 and LC90 values of 1.13 and 2.85 ppm respectively, against third-instar larvae of Cx. quinquefasciatus at 24 h. In control, the larvae exhibited normal movement. Tiliamosine exhibited 91% ovicidal activity at 2.0 ppm concentration after 120 h post-treatment. Lowest concentration of tiliamosine (0.5 ppm) showed 19% egg mortality. Histopathology study of the compound-treated larvae showed serious damage on the larval midgut cells. The treated larvae showed restless movement which was different from that of the control larvae. The larvae exhibited malformation in development. The compound tiliamosine was harmless to non-target organisms P. reticulata and Dragon fly nymph at tested concentrations. The compound was highly active and inhibited AChE in a concentration-dependent manner. Computational analysis of the tiliamosine had strong interaction with AChE1 of Cx. quinquefasciatus. This report clearly suggests that the isolated compound can be used as an insecticide to control mosquito population and thus prevent the spread of vector-borne diseases.

Insecticidal efficacy of the essential oil of jambú (Acmella oleracea (L.) R.K. Jansen) cultivated in central Italy against filariasis mosquito vectors, houseflies and moth pests.

ETHNOPHARMACOLOGICAL RELEVANCE: Acmella oleracea (L.) R.K. Jansen (Compositae), well-known as jambú, is a medicinal herb of pungent taste, native to Brazil but cultivated in different parts of the world due to its aromatic and pharmacological properties. In folk medicine, the plant has been used against parasites and to combat insects and mites. No data are available on the insecticidal activity of jambú essential oil. AIM OF THE STUDY: To test the jambú essential oil obtained from A. oleracea cultivated in central Italy against the filariasis vector, Culex quinquefasciatus, the Egyptian cotton worm, Spodoptera littoralis, and the housefly, Musca domestica. MATERIALS AND METHODS: The chemical composition of the essential oil was achieved by GC-FID and GC-MS analyses. Acute toxicity experiments were conducted on larvae of the filariasis vector C. quinquefasciatus and S. littoralis and adults of M. domestica to determine the LC50(LD50) and LC90(LD90) values of the oil, along with the positive control, α-cypermethrin. RESULTS: (E)-caryophyllene (20.8%), ß-pinene (17.3%), myrcene (17.1%) and caryophyllene oxide (10.0%) were the major volatile constituents. Interestingly, the oil contained little amounts (3.9%) of the insecticidal spilanthol. Jambú essential oil exerted relevant effects on C. quinquefasciatus (LC50 = 42.2 mg L-1, LC90 = 73.6 mg L-1) and S. littoralis 3rd instar larvae (LD50 = 68.1 µg larva-1, LD90 = 132.1 µg larva-1). High acute toxicity was also detected testing the jambú oil against adult females of M. domestica, achieving a LD50 value of 44.3 µg adult-1 and a LD90 value of 87.5 µg adult-1. CONCLUSIONS: Taken together our data support the traditional use of jambú as an insecticidal agent and represent the scientific basis for the industrial exploitation of the essential oil in the fabrication of green insecticides.

Larvicidal activity of some medicinal plant extracts against filariasis fever mosquito, Culex quinquefasciatus (Say.) (Diptera: Culicidae).

OBJECTIVES: The present study was aimed to find the larvicidal activity of petroleum ether, chloroform, acetone and methanolic extracts of Dichanthium foveolatum (Del.) Roberty, Leptochloa uniflora Hochst, Pancratium triflorum Roxb and Molineria trichocarpa (Wight) N.P.Balakr against Culex quinquefasciatus. METHODS: The larvicidal potential of selected plant extracts were determined against 4th instar larvae of C. quinquefasciatus with various concentrations viz., 50, 100, 150, 200 and 250 mg/ml. The mortality counts were made after 24 h of incubation and LC50 values were calculated. RESULTS: Chloroform extracts of studied plants were showed highest larvicidal activity with remarkable irritant against the larva of C. quinquefasciatus. Highest larvicidal activity was observed in the chloroform extract of D. foveolatum against the larva of C. quinquefasciatus with LC50 = 277.03 mg/ml. The larvicidal activity of the studied plants as follows chloroform extract of D. foveolatum (LC50 = 277.03 mg/ml) >L. uniflora (LC50 = 300.56 mg/ml) >M. trichocarpa (LC50 = 306.60 mg/ml) >P. triflorum (LC50 318.42 mg/ml). The larvicidal potential of P. triflorum was as follows Chloroform > acetone > methanol > petroleum ether. The larvicidal activities of L. uniflora and M. trichocarpa were as follows Chloroform > petroleum ether > acetone > methanol respectively. The larvicidal potentials of D. foveolatum was as follows Chloroform > methanol > acetone > petroleum ether. CONCLUSION: The chloroform extract of D. foveolatum find use as broad-spectrum larvicidal agent in the near future. It is hoped that more work would be undertaken to evaluate the utility of these plant extracts for field applications considering the promising leads given by the present study.

Novel Findings of Anti-Filarial Drug Target and Structure-Based Virtual Screening for Drug Discovery.

Lymphatic filariasis and onchocerciasis caused by filarial nematodes are important diseases leading to considerable morbidity throughout tropical countries. Diethylcarbamazine (DEC), albendazole (ALB), and ivermectin (IVM) used in massive drug administration are not highly effective in killing the long-lived adult worms, and there is demand for the development of novel macrofilaricidal drugs affecting new molecular targets. A Ca2+ binding protein, calumenin, was identified as a novel and nematode-specific drug target for filariasis, due to its involvement in fertility and cuticle development in nematodes. As sterilizing and killing effects of the adult worms are considered to be ideal profiles of new drugs, calumenin could be an eligible drug target. Indeed, the Caenorhabditis elegans mutant model of calumenin exhibited enhanced drug acceptability to both microfilaricidal drugs (ALB and IVM) even at the adult stage, proving the roles of the nematode cuticle in efficient drug entry. Molecular modeling revealed that structural features of calumenin were only conserved among nematodes (C. elegans, Brugia malayi, and Onchocerca volvulus). Structural conservation and the specificity of nematode calumenins enabled the development of drugs with good target selectivity between parasites and human hosts. Structure-based virtual screening resulted in the discovery of itraconazole (ITC), an inhibitor of sterol biosynthesis, as a nematode calumenin-targeting ligand. The inhibitory potential of ITC was tested using a nematode mutant model of calumenin.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.

Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) extracts exhibit strong larvicidal activity against mosquito vectors of malaria, dengue fever, and filariasis.

Vector-borne diseases transmitted by mosquitoes cause globally important diseases such as malaria, dengue fever, and filariasis. The incidence of these diseases can be reduced through mosquito control programs but these control programs currently rely on synthetic insecticides that can impact the environment, and has selected widespread mosquito resistance. Environment friendly and biodegradable natural insecticides discovered in plants offer an alternative approach to mosquito control. Here, we investigated extracts from root or aerial parts of Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) against the early 4th instar larvae of Anopheles stephensi (malaria vector), Aedes aegypti (dengue fever vector), and Culex quinquefasciatus (filariasis vector). The root and aerial parts extracts of A. absinthium and C. intybus at 200, 100, 50, 25 and 12.5 ppm caused significant mortality of the tested mosquito species. Root extracts exhibited higher larvicidal activity that aerial part extracts. The highest larvicidal activity was recorded in methanol extract of roots of C. intybus with LC50 = 66.16, 18.88 and LC¬90 = 197.56, 107.16 ppm for An. stephensi; LC50 = 78.51, 40.15 and LC90 = 277.31, 231.28 ppm for Ae. aegypti and LC50 = 103.99, 64.56 and LC¬90 = 314.04, 247.54 ppm for Cx. quinquefasciatus. These results reveal potent mosquito larvicidal activity against vectors of malaria, dengue fever, and filariasis is present in extracts of chicory and wormwood.

Validation of ultrasound bioimaging to predict worm burden and treatment efficacy in preclinical filariasis drug screening models.

Filariasis is a global health problem targeted for elimination. Curative drugs (macrofilaricides) are required to accelerate elimination. Candidate macrofilaricides require testing in preclinical models of filariasis. The incidence of infection failures and high intra-group variation means that large group sizes are required for drug testing. Further, a lack of accurate, quantitative adult biomarkers results in protracted timeframes or multiple groups for endpoint analyses. Here we evaluate intra-vital ultrasonography (USG) to identify B. malayi in the peritonea of gerbils and CB.17 SCID mice and assess prognostic value in determining drug efficacy. USG operators, blinded to infection status, could detect intra-peritoneal filarial dance sign (ipFDS) with 100% specificity and sensitivity, when >5 B. malayi worms were present in SCID mice. USG ipFDS was predictive of macrofilaricidal activity in randomized, blinded studies comparing flubendazole, albendazole and vehicle-treated SCID mice. Semi-quantification of ipFDS could predict worm burden >10 with 87-100% accuracy in SCID mice or gerbils. We estimate that pre-assessment of worm burden by USG could reduce intra-group variation, obviate the need for surgical implantations in gerbils, and reduce total SCID mouse use by 40%. Thus, implementation of USG may reduce animal use, refine endpoints and negate invasive techniques for assessing anti-filarial drug efficacy.

Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi.

Antifilarial potential of three medicinal plants namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa was explored using Setaria cervi, a bovine filarial parasite at concentrations of 2.5, 5 and 10mg/ml. Amongst all the extracts, methanol extract of T. bellerica showed highest macrofilaricidal activity i.e. 84.63±1.11 at 10mg/ml in MTT reduction assay with IC50 value of 2.7mg/ml. which was better than the standard DEC i.e. 79.22±3.1% at 10mg/ml with IC50 value 2.84mg/ml. Other plant extracts showed mild in vitro macrofilaricidal activity. T. bellerica methanol extract exhibited significant GST activity of 18.86±0.21 and 12.83±0.03µM/ml/min at 5 and 10mg/ml with percentage inhibition value of 73.96% and 82.29% respectively. DEC showed GST activity value of 40.03±4.14 and 21.48±6.44µM/ml/min with percentage inhibition value of 21.76% and 58.01% at 5 and 10mg/ml respectively. Thus, methanol extract of leaves of T. bellerica exhibited highly significant antifilarial potential and needs detailed analysis.

Combinations of registered drugs reduce treatment times required to deplete Wolbachia in the Litomosoides sigmodontis mouse model.

Filarial parasites can be targeted by antibiotic treatment due to their unique endosymbiotic relationship with Wolbachia bacteria. This finding has led to successful treatment strategies in both, human onchocerciasis and lymphatic filariasis. A 4-6 week treatment course using doxycycline results in long-term sterility and safe macrofilaricidal activity in humans. However, current treatment times and doxycycline contraindications in children and pregnant women preclude widespread administration of doxycycline in public health control programs; therefore, the search for shorter anti-wolbachial regimens is a focus of ongoing research. We have established an in vivo model for compound screening, using mice infected with Litomosoides sigmodontis. We could show that gold standard doxycycline treatment did not only deplete Wolbachia, it also resulted in a larval arrest. In this model, combinations of registered antibiotics were tested for their anti-wolbachial activity. Administration of rifamycins in combination with doxycycline for 7 days successfully depleted Wolbachia by > 2 log (>99% reduction) and thus resulted in a significant reduction of the treatment duration. Using a triple combination of a tetracycline (doxycycline or minocycline), a rifamycin and a fluoroquinolone (moxifloxacin) led to an even greater shortening of the treatment time. Testing all double combinations that could be derived from the triple combinations revealed that the combination of rifapentine (15mg/kg) and moxifloxacin (2 x 200mg/kg) showed the strongest reduction of treatment time in intraperitoneal and also oral administration routes. The rifapentine plus moxifloxacin combination was equivalent to the triple combination with additional doxycycline (>99% Wolbachia reduction). These investigations suggest that it is possible to shorten anti-wolbachial treatment times with combination treatments in order to achieve the target product profile (TPP) requirements for macrofilaricidal drugs of no more than 7-10 days of treatment.

Exploration of antifilarial activity of gold nanoparticle against human and bovine filarial parasites: A nanomedicinal mechanistic approach.

The present work seeks to explore the antifilarial activity of biopolymer functionalized gold nanoparticles (AuNPs) against human filarial parasite (Wuchereria bancrofti) through Nrf2 signaling for the first time. A natural polymer, chitosan is used along with Terminalia chebula extract to synthesize AuNPs following the principles of green chemistry. The probable mode of action of AuNPs as filaricidal agent has been investigated in detail using model filarial parasite, Setaria cervi (bovine parasite). Biopolymers inspired AuNPs exhibit superior antifilarial activity against both human and bovine filarial parasites, and are able to induce oxidative stress and apoptotic cell death in filarial parasites mediated through mitochondria. AuNPs also alter the Nrf2 signaling. In addition, the synthesized nanomaterials appear to be nontoxic to mammalian system. Thus the present mechanistic study, targeting human filarial parasites, has the potential to increase the therapeutic prospects of AuNPs to control lymphatic filariasis in the upcoming days.

Toxicity studies of nanofabricated palladium against filariasis and malaria vectors.

The present study was carried out to establish the biofabrication of palladium nanoparticles (PdNPs) using the plant leaf extract of Tinospora cordifolia Miers and its toxicity studies on the larvae of filariasis vector, Culex quinquefasciatus Say and malaria vector, Anopheles subpictus Grassi. The biofabricated PdNPs were characterized by using UV-visible spectrum, FTIR, XRD, FESEM, EDX and HRTEM. HRTEM confirmed the PdNPs were slightly agglomerated and spherical in shape and the average size was 16 nm. Gas chromatography and mass spectrometry analysis result revealed that the major constituent present in the T. cordifolia leaf extract is 2,4-di-tert-butylphenol (31.79%) whereas the minor compounds are 1-hexadecanol (7.97%), 1-octadecanol (7.70%), 1-eicosanol (6.85%), behenic alcohol (5.36%), 1-tetradecene (6.22%), cyclotetradecane (6.23%), 1-hexadecene (7.97%), 1-octadecene (7.70%), 1-eicosene (6.85%), and 1-docosene (5.36%). T. cordifolia leaf extract exhibited the larvicidal activity against the fourth instar larvae of C. quinquefasciatus and A. subpictus with the values of LC50 = 59.857 and 54.536 mg/L; LC90 = 113.445 and 108.940 mg/L, respectively. The highest toxicity was observed in the biofabricated PdNPs against the fourth instar larvae of C. quinquefasciatus and A. subpictus with the values of LC50 = 6.090 and 6.454 mg/L; LC90 = 13.689 and 13.849 mg/L, respectively. Concerning non-target effects, Poecilia reticulata were exposed to PdNPs for 24 h and did not exhibit any noticeable toxicity. Overall, our findings strongly suggest that PdNPs is a perfect ecological and inexpensive approach for the control of filariasis and malaria vectors.

Traditional herbal remedies and dietary spices from Cameroon as novel sources of larvicides against filariasis mosquitoes?

In Cameroon, many dietary spices are used by traditional healers to cure several diseases such as cancer and microbial infections. Aframomum daniellii, Dichrostachys cinerea and Echinops giganteus are Cameroonian spices widely used as flavourings and as food additives. Moreover, they are traditionally herbal remedies employed to treat several diseases, as well as to control populations of insect pests. In this research, we analysed the chemical composition of A. daniellii, D. cinerea and E. giganteus essential oils and we evaluated their larvicidal potential against larvae of the filariasis and West Nile virus vector Culex quinquefasciatus. The essential oils were obtained from different plant parts by hydrodistillation and their composition was analysed by GC-MS. The three spices exhibited different volatile chemical profiles, being characterized by 1,8-cineole, sabinene and ß-pinene (A. daniellii), geraniol and terpinen-4-ol (D. cinerea), and silphiperfol-6-ene and presilphiperfolan-8-ol (E. giganteus). Results showed that the highest larvicidal toxicity on Cx. quinquefasciatus was exerted by D. cinerea essential oil (LC50 = 39.1 µL L-1), followed by A. daniellii (pericarp essential oil: LC50 = 65.5 µL L-1; leaves: LC50 = 65.5µL L-1; seeds: LC50 = 106.5µL L-1) and E. giganteus (LC50 = 227.4 µL L-1). Overall, the chance to use the D. cinerea essential oil against Cx. quinquefasciatus young instars seems promising, since it is effective at moderate doses and could be an advantageous alternative to build newer mosquito control tools.

Endothelial cells release soluble factors that support the long-term survival of filarial worms in vitro.

The inability to maintain filarial nematodes in long-term in vitro culture greatly limits research into the basic biology of these parasites and hinders in vitro screening of novel anti-filarial agents. In this study, we sought to characterize nutrients that promote the long-term survival of filarial worms in vitro. Using microfilariae (MF) obtained from gerbils infected with Litomosoides sigmodontis, a filarial parasite of rodents, we found that Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) resulted in MF survival of only 5 days. However, co-culturing MF with a mouse endothelial cell line (EOMA) enabled survival for 40 days. Culturing EOMA cells in transwell plates extended MF survival to the same degree as direct co-culture, suggesting that the factors microfilariae require are soluble in nature. Heat inactivation of EOMA conditioned media at 56 °C reduced MF survival by approximately 50%, and heat inactivation at 100 °C reduced survival to 3 days, demonstrating that both heat labile and heat stable factors are involved. EOMA cells require FBS to produce these factors, as conditioned media collected from EOMA cells grown in the absence of FBS failed to prolong survival. The removal of lipids also abrogated survival, indicating MF are likely utilizing lipid factors released by EOMA cells. Dialysis experiments demonstrate that at least some of the required factors are between 0.1 and 1 kDa in size. Importantly, L. sigmodontis adult worms also show significantly extended survival when cultured in EOMA conditioned media. Together, these results suggest that EOMA-produced factors include lipid-containing molecules, heat labile molecules (likely a protein), and micronutrients between 0.1 and 1 kDa in size. These studies have established a cell-free approach to maintaining MF and adult stage filarial worms in long-term in vitro culture and have taken important steps towards biochemically characterizing host-derived nutrients required for parasite survival.

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections.

BACKGROUND: A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. METHODOLOGY/ PRINCIPAL FINDINGS: Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. CONCLUSIONS: The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites.