Crude protein fraction with high thioredoxin reductase (TrxR) enzyme activity from filarial parasite Setaria cervi counters lipopolysaccharide (LPS)-induced inflammation in macrophages.

Host-parasite interaction has always been an area of interest to the parasite biologists. The complex immune interactions between the parasite and/or the parasite-derived products with the host immune cells determine the fate of the disease biology. Parasitic organisms are widely equipped with a vast array of protective machineries including antioxidant enzymes to withstand the hostile condition inside the host body. The reactive oxygen species (ROS) generated inside the host as a result of parasitic intervention can be endured by the parasite by their own tools to ensure their survival. One such antioxidant enzyme in the filarial parasite that plays a significant role in redox homeostasis, survivability and disease progression is the thioredoxin reductase (TrxR). Herein, we have projected a crude lysate of the bovine filarial parasite Setaria cervi enriched with high TrxR enzyme activity has the capacity to downregulate lipopolysaccharide (LPS)-induced inflammatory macrophages. TrxR-mediated inhibition of the TLR4-NF-κB axis resulting into downregulation of the pro-inflammatory cytokines with concomitant upregulation of the anti-inflammatory cytokines supports the filarial parasite to produce an anti-inflammatory milieu which ultimately promotes worm survivability inside the host and pathogenesis.

Inhibition of thioredoxin reductase (TrxR) triggers oxidative stress-induced apoptosis in filarial nematode Setaria cervi channelized through ASK-1-p38 mediated caspase activation.

Inhibition of an imperative antioxidant enzyme with subsequent death is a victorious and widely accepted strategy to combat various infectious diseases. Among different antioxidant enzymes, thioredoxin reductase (TrxR) is an exclusive one. Studies have revealed that direct inhibition of TrxR by different classes of chemical moieties promptly results in the death of an organism. Especially the structural as well as biochemical modifications of the enzyme upon inhibition project serious threat towards the subject organism. Herein, an attempt was made to inhibit TrxR of filarial species by administering Auranofin, 1 chloro 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo dithioperoxo(thioate) derivative (4a). Our study has revealed that inhibition of TrxR resulted in the induction of the classical CED pathway of apoptosis along with the intrinsic and extrinsic pathways of apoptosis (Caspase mediated) routed through the ASK-1/p38 axis. Druggability analysis of filarial TrxR for the selected compounds was performed in silico through molecular docking studies. Therefore, this study attempts to decipher the mechanism of apoptosis induction following TrxR inhibition. The safety of those four compounds in terms of dose and toxicity was taken under consideration. Thitherto, the mechanism of TrxR mediated initiation of cell death in filarial parasite has remained undercover, and therefore, it is a maiden report on the characterization of apoptosis induction upon TrxR inhibition which will eventually help in generating effective antifilarial drugs in the future.

Senna plant generates reactive oxygen species (ROS) and induces apoptosis in Hymenolepis diminuta.

Like mammalian cells, helminth parasites are equipped with an array of enzymatic anti-oxidant system which has an adaptive strategy to cope up with several conditions of stress that arise from host immune response or drug treatment. Earlier, we had reported that three species of Senna, viz. S. alata, S. alexandrina and S. occidentalis leaf extracts caused severe morphological and biochemical alterations in the zoonotic parasite Hymenolepis diminuta. To understand whether the leaf extracts of the three species of Senna have any effect on the enzymatic anti-oxidant system in H.diminuta or not, the present study was investigated on the mechanism of action of these leaf extracts on the anti-oxidant system of the parasite. The viability of the parasite was assessed by MTT reduction assay, chromatin condensation through Hoechst staining of tissue and DNA fragmentation assay, and the oxidative enzymes of the parasite were estimated biochemically. Activity of superoxide dismutase, catalase, glutathione S- transferase and glutathione peroxidase were found to be increased in all the treated parasites from that of the control, with S. alata showed the highest increased amongst the three plant species in all the enzymes, at 331.0 %, 215.4 %, 85.4 % and 65.5 % respectively. Upliftment of apoptotic protein CED-3, CED-4 and EGL-1 and down regulation of anti-apototic protein CED-9 was visualised in all treated paraites. The redox imbalance triggered by these leaf extracts resulted in the activation of apoptotic pathway that led to death of the parasite. Our results demonstrated that the leaf extracts of the three Senna plant species could open new insight for an affordable natural anthelmintic with high efficacy and less toxicity.

Wuchereria bancrofti filaria activates human dendritic cells and polarizes T helper 1 and regulatory T cells via toll-like receptor 4.

Interaction between innate immune cells and parasite plays a key role in the immunopathogenesis of lymphatic filariasis. Despite being professional antigen presenting cells critical for the pathogen recognition, processing and presenting the antigens for mounting T cell responses, the dendritic cell response and its role in initiating CD4+ T cell response to filaria, in particular Wuchereria bancrofti, the most prevalent microfilaria is still not clear. Herein, we demonstrate that a 70 kDa phosphorylcholine-binding W. bancrofti sheath antigen induces human dendritic cell maturation and secretion of several pro-inflammatory cytokines. Further, microfilarial sheath antigen-stimulated dendritic cells drive predominantly Th1 and regulatory T cell responses while Th17 and Th2 responses are marginal. Mechanistically, sheath antigen-induced dendritic cell maturation, and Th1 and regulatory T cell responses are mediated via toll-like receptor 4 signaling. Our data suggest that W. bancrofti sheath antigen exploits dendritic cells to mediate distinct CD4+ T cell responses and immunopathogenesis of lymphatic filariasis.

Quinolone-fused cyclic sulfonamide as a novel benign antifilarial agent.

Search of potent antifilarial drugs has been a major thrust area in tropical medicine research over the decades. Herein, we report 4,7-dimethyl-3,4,7,8-tetrahydro-3λ6-[1,2]thiazino[4,3-f]quinoline-3,3,8-trione (8l) as a new class of antifilarial agent which is extremely potent, with lethality against all the developmental stages (oocyte, microfilaria and adult) of the filarial parasite Setaria cervi. Molecular investigation on its mode of action revealed that 8l is a typical inducer of reactive oxygen species that triggers oxidative stress inside the filarid and further signals induction of apoptosis by activating both intrinsic and extrinsic pathways. Moreover, 8l is also active against Wolbachia, the essential endosymbiont of several human infectious filarids. Selective toxicity against filarial parasites and non-toxic nature in rat model were found as unique traits of 8l to be a future medicine. Taken en masse, this maiden report on a novel quinolone fused cyclic sulfonamide presents a promising therapeutic lead for lymphatic filariasis in future.

Thioredoxin reductase from the bovine filarial parasite Setaria cervi: Studies on its localization and optimization of the extraction.

Exploration of novel drug targets has been the major thrust area in filarial research. In this regard, identification and characterization of oxidative enzymes that play pivotal role in the survival of filarial parasite inside host are of immense importance. In this study, we are reporting the presence of an important redox regulatory enzyme, thioredoxin reductase (TrxR) in the bovine filarial parasite Setaria cervi. TrxR was found to be exists throughout the developmental stages viz. oocyte, microfilaria and adult of the parasite. Since further studies on this enzyme require adequate quantity, influential extraction parameters were optimized statistically using response surface methodology (RSM) employing a seven factors based Box-Behnken design matrix. ANOVA analysis revealed the relative importance of each parameter and a regression equation was eventually developed that could predict the specific activity (SA) of TrxR. Finally the optimized extraction conditions predicted by RSM was 6.1ml of 61.86mM buffer, pH 6.0, with extraction temperature 39.96°C for 180min in addition to 450rpm agitation and 20µl/ml of protease inhibitor. Therefore this study is going to be the maiden report depicting the identity of TrxR in filarial parasite and the optimized extraction conditions for its isolation with better kinetic efficiency.

Development of chitosan based gold nanomaterial as an efficient antifilarial agent: A mechanistic approach.

The gold nanoparticles (AuNPs) have been synthesized biogenically by using black pepper (Piper nigrum) extract according to the principles of green chemistry in presence and absence of a biopolymer, chitosan. A comprehensive study (up to cellular level) on the antifilarial (against Setaria cervi) activity of AuNPs has been made for the first time with a view to use it clinically. The bioactivity of biopolymer capped biogenic AuNP increases significantly compared to simple biogenic AuNP. The biopolymer plays an important role in inspiring AuNP through its inherent positive charges and hydrophobicity. The developed nanomaterial boosts the production of ROS (reactive oxygen species) and misbalances the antioxidant parameters of parasites such as GSH, GST, GPx, SOD and catalase. The produced ROS ultimately induces oxidative stress, which leads to apoptotic cell death in filarial worms. The synthesized nanomaterials exhibit negligible toxicity towards human PBMCs. The present study may serve as a fruitful platform to explore biopolymer capped gold nanoparticles as efficient antifilarial therapeutics.

Metabolic Inhibitors as Antiparasitic Drugs: Pharmacological, Biochemical and Molecular Perspectives.

BACKGROUND: Human diseases caused by the infectious parasites have been one of the major problems throughout the evolutionary journey. Protozoan and metazoan parasitic infections result in a large number of deaths, disabilities and socio-economic loss worldwide to date. Despite the best efforts for developing suitable antiparasitics, these infections take a massive toll on human health. The prevalence of emerging resistance to the existing drugs, lack of efficacy and toxic side effects are as added complications. Being enlisted under 'neglected' category, serious diseases like leishmaniasis, filariasis, trypanosomiasis etc. have failed to draw attention of the governments as well as the pharmaceutical companies. Thus, target specific as well as cost-convenient therapy needs to be employed for the treatment of these diseases and selective targeting of metabolic pathways appears to be the most promising mean. METHODS: In this context, quality works have been explored for screening either anti-metabolic drugs or selective targets in different groups of parasites. Moreover, complete genome sequencing and metabolomic profiling have provided the initiatives to search for new lethal targets in parasites. RESULTS: New metabolic targets are being reported from different organelles and other sub-cellular compartments of parasites such as mitochondrion, kinetoplast, apicoplast, glycosome, hydrogenosome, acidocalcisome, plasma membrane, cytoskeleton, etc. Herein, unique findings achieved in identifying new antimetabolic drugs or targets and studying their molecular mode of actions have been reviewed by incorporating existing and upcoming approaches. CONCLUSION: Considering the alarming scenario of diseases caused by parasites globally, this paper provides a comprehensive review to the scientific community on the development of novel interventions based on metabolic targets to combat the challenges posed by parasites.

Phenolics and Terpenoids; the Promising New Search for Anthelmintics: A Critical Review.

Ailments caused by helminth parasites are global causing different types of clinical complications with permanent and long term morbidity in humans. Although huge advances have been made in medical sciences the effectiveness of available anthelmintics are still quite limited. Starting from the 50's, most importance was given to synthetic compounds for developing remedies from them, however, the traditional knowledge of medicine of different countries continued to provide us clues against this widespread health problem. Natural products or structural analogs with diverse structures are always been the major sources for discovering new therapeutics and in recent past different active compounds have also been identified form these plant sources having anthelmintic properties. Although compounds of diverse chemical nature and classes were identified, most active ones belong to either phenol or terpene in broad chemical nature. The mechanism of action of these phytotherapeutics is usually multi-targeted and can act against the helminth parasites through diverse spectrum of activities. In this review we summarized the effective anthelmintics belong to either phenolics or terpenoids and highlighted the major way of their effectiveness. This also highlights the recent development of new therapeutic strategies against helminth parasites in the light of recent advances of knowledge. In addition, developing efficient strategies to promote apoptosis and disturbing redox status in them by natural products can provide us a clue in antifilarial drug developmental research and crucial unmet medical need.

Ginger extract ameliorates phosphamidon induced hepatotoxicity.

Organophosphorus (OP) compounds commonly used as pesticides in agriculture cause serious health problems to living beings. The present study enumerates the ameliorating effect of ginger extract (GE) against phosphamidon (PHO, an organophosphorus insecticide) induced hepatotoxicity. GE was prepared from dried ginger and characterized for compound profile and antioxidant activity. Eight groups of albino rats (n = 6) were treated with 1/5th lethal dose of PHO for 5-20 days. Out of the treated 8 groups, 4 were simultaneously fed with GE (1 mg/kg body wt.) along with PHO. Alterations in the levels of hepatocellular oxidative stress (OS) markers in the treated groups indicated an enhanced generation of reactive oxygen species (ROS) and oxidative stress (OS). Upregulation of apoptotic markers, DNA fragmentation and appearance of apoptotic nuclei suggested induction of apoptosis in the liver cell that was found to be attenuated after GE treatment. Moreover, no toxicity and mortality was observed up to 100 mg/kg dose of GE for 30 days in the rat model studied. Thus, GE can be considered as an effective, economical and safe extract to circumvent PHO-induced hepatotoxicity.

Diospyros perigrena bark extract induced apoptosis in filarial parasite Setaria cervi through generation of reactive oxygen species.

CONTEXT: Lymphatic filariasis is a major neglected tropical disease. Diospyros perigrena Gurke (Ebenaceae) was selected for antifilarial chemotherapy because of unavailability of proper medicine. In India, different parts of this plant were used for the treatment of diabetes, diarrhea, dysentery, cholera, mouth ulcers, and wounds. OBJECTIVE: The present study was undertaken to access antifilarial potential and mechanism of action of n-butanol extract (NBE) of D. perigrena stem bark on Setaria cervi Rudolphi (Onchocercidae). MATERIALS AND METHODS: In vitro efficacy and apoptotic mechanism were evaluated by Hoechst, TUNEL, DNA fragmentation assay, pro- and anti-apoptotic gene expression in NBE (250, 125, 62.5, 31.25, and 15.6 µg/ml)-treated S. cervi after 24 h of incubation. Reactive oxygen species (ROS) up-regulation was also determined by GSH, GST, SOD assays, and super oxide anion level. RESULTS: Significant in vitro antifilarial activity of NBE was found 50% inhibitory concentration (IC50): adult = 57.6 µg/ml, microfilariae (mf) = 56.1 µg/ml, and lethal dose (LD100) in mf is 187.17 µg/ml) after 24 h of treatment. NBF-induced apoptosis was proved by Hoechst, TUNEL, RT-PCR, and Western blot method. NBF (250 µg/ml) decreased the level of GSH (17.8%) and GST (65.4%), increased SOD activity (1.42-fold) and super oxide anion production (1.32-fold) in the treated parasite which culminated into ROS up-regulation. DISCUSSION AND CONCLUSION: NBE induced apoptosis in different life cycle stages of S. cervi. In future, a detailed study of NBF will give us a novel antifilarial compound which will be used for antifilarial chemotherapy.

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria.

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.

Antifilarial effect of ursolic acid from Nyctanthes arbortristis: molecular and biochemical evidences.

A bio-assay guided fractionation and purification approach was used to examine in vitro antifilarial activities of the crude methanolic extract of Nyctanthes arbortristis as well as fractions and isolated compound. From ethyl-acetate fraction we isolated and identified a triterpenoid compound which has been characterized as ursolic acid (UA) by HPLC and NMR data. We are reporting for the first time isolation and identification of UA from the leaves of N. arbortristis. The crude extract and UA showed significant micro- as well as macrofilaricidal activities against the oocyte, microfilaria and adult of Setaria cervi (S. cervi) by dye exclusion test and MTT reduction assay. Significant microfilaricidal activity of UA was further proved against mf of W. bancrofti by viability assay. The findings thus provide a new lead for development of a suitable filaricide from natural products. The molecular mechanism of UA was investigated by performing TUNEL, Hoechst staining, Annexin V-Cy3, flow cytometric analysis and DNA fragmentation assay. Differential expressions of pro- and anti-apoptotic genes were observed at the transcription and translational levels in a dose-dependent manner. Depletion in the worm GSH level and elevation in the parasite GST, SOD and super oxide anion indicated the generation of ROS. In this investigation we are reporting for the first time that UA acts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants like GSH, GST and SOD of S. cervi.

Molecular evidence of curcumin-induced apoptosis in the filarial worm Setaria cervi.

Curcumin (diferuloyl methane) is a major curcuminoid from Curcuma longa that exhibits various pharmacological effects and has shown multiple beneficial activities. Our understanding of its anticarcinogenic and other activities occurring through curcumin-induced apoptosis in several cancer cells has greatly expanded in recent years. Lymphatic filariasis is a worldwide health problem causing global disability in humans and is caused by filarial nematodes. Development of efficient strategies to promote programmed cell death in filarial worms remains a key challenge for anti-filarial drug developing research and a crucial unmet medical need. In this study, we have taken molecular and biochemical approaches toward understanding the molecular basis for curcumin-mediated anti-filarial activity in the filarial nematode Setaria cervi. Results of MTT assay showed that curcumin causes a significant reduction in viability of Mf and adults and thus acts as a potent macro- and micro-filaricidal agent. Hoechst staining, TUNEL staining, showed several apoptotic nuclei in different parts of curcumin-treated adults. At 25 µM concentration it showed chromosomal DNA fragmentation in adult worms. Our results indicate that curcumin decreases protein and mRNA expression levels of anti-apoptotic gene ced-9 and enhances both the levels of pro-apoptotic genes ced-3 and ced-4 in a dose-dependent manner. All these observations ascertained the apoptogenicity of curcumin at a minimum concentration of 50 µM in this filarial worm. Furthermore, we showed that curcumin causes depletion of parasitic glutathione level, enhances the activities of glutathione S-transferase and superoxide dismutase and stimulates rapid generation of reactive oxygen species (ROS). Here, we present molecular evidence on curcumin-induced apoptosis in the filarial nematode S. cervi with probable involvement of ROS in a caspase-dependent manner.

Evidence for Wolbachia symbiosis in microfilariae of Wuchereria bancrofti from West Bengal, India.

Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.