CpG enhances the immunogenicity of heterologous DNA-prime/protein-boost vaccination with the heavy chain myosin of Brugia malayi in BALB/c mice.

The recombinant heavy chain myosin of Brugia malayi (Bm-Myo) has earlier been reported as a potent vaccine candidate in our lab. Subsequently, we further enhanced its efficacy employing heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) immunization approach that produced superior immune-protection than protein or DNA vaccination. In the present study, we evaluated the efficacy of heterologous prime boost vaccination in combination with CpG, synthetic oligodeoxynucleotides (ODN) adjuvant in BALB/c mice. The results showed that CpG/Myo-pcD+Bm-Myo conferred 84.5 ± 0.62% protection against B. malayi infective larval challenge which was considerably higher than Myo-pcD+Bm-Myo (75.6 ± 1.10%) following immunization. Although, both the formulations of immunization elicited robust production of specific IgG antibody and their isotypes (IgG1, IgG2a, IgG2b, and IgG3); however, CpG/Myo-pcD+Bm-Myo predominantly enhanced the level of IgG2a suggesting Th1 biased immune response in presence of CpG. Furthermore, spleen isolated from mice that immunized with CpG/Myo-pcD+Bm-Myo had greater accumulation of CD4+, CD8+, and CD19+ B cells and there was an augmented expression of co-stimulatory molecules CD40, CD86 on host dendritic cells (DCs). In contrast to Myo-pcD+Bm-Myo group, the splenocytes of CpG/Myo-pcD+Bm-Myo immunized mice developed comparatively higher pro-inflammatory cytokines IL-2 and IFN-γ leaving anti-inflammatory cytokine levels unchanged. Moreover, CpG formulation also upregulated the RNA expression of IL-12 and TNF-α in spleenocytes. The current findings suggest that the use of CpG would be more advantageous as an adjuvant predominantly in DNA/protein prime boost vaccine against Bm-Myo and presumably also for filarial infection.

Molecular characterization of novel immunodominant molybdenum cofactor biosynthesis protein C1 (Rv3111) from Mycobacterium tuberculosis H37Rv.

BACKGROUND: In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature. METHODS: We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques. RESULTS: We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. CONCLUSION: Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. GENERAL SIGNIFICANCE: This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.

Regulatory T-cell neutralization in mice during filariasis helps in parasite clearance by enhancing T helper type 17-mediated pro-inflammatory response.

Lymphatic filariasis leads to profound impairment of parasite-specific T helper type 1 (Th1) and Th2 immune responses and significantly increases the expression of regulatory networks and regulatory effectors like transforming growth factor-ß, CD25, cytotoxic T-lymphocyte antigen 4, glucocorticoid-induced tumour necrosis factor receptor (GITR) and regulatory T (Treg) cells, which together play an important role in immunosuppression. While Treg cells suppress the activity of effector cells, monocyte dysfunction, characterized by an alternatively activated immunoregulatory phenotype, is one hypothesis that explains the lack of an antigen-specific T-cell response in infected individuals. In the present study, we administered neutralizing antibodies against the Treg cell-associated markers CD25 and GITR and observed its effects on filaria-induced immunosuppression. Our results show that administration of anti-CD25 and anti-GITR in infected animals not only arrested the accumulation of Treg cells and reduced arginase activity, but also led to an increase in the percentages of Th17 cells in the secondary lymphoid organs of mice. Elevated levels of interferon-γ and decreased levels of interleukin-10 were also noted in the culture supernatants of mouse splenocytes that were treated with neutralizing antibodies. Furthermore, treatment with neutralizing antibodies enhanced the expression of inducible nitric oxide synthase on host macrophages and CD40 on host dendritic cells with concomitant decreased expression of alternative activation markers Arg1, Ym1 and Fizz1, which together lead to reduced parasite burden in treated animals. In summary, administration of neutralizing antibodies helps in breaking the regulatory network in mice and limits parasite-induced immunosuppression at the earliest host-parasite interface.

Subcutaneously Administered Ultrafine PLGA Nanoparticles Containing Doxycycline Hydrochloride Target Lymphatic Filarial Parasites.

Systemic chemotherapeutic targeting of filarial parasites is unfocused due to their deep seated location in lymphatic vessels. This warrants a prolonged dosing regimen in high doses for an anthelmintic like doxycycline hydrochloride (DOX). In order to provide an alternative, we have constructed ultrafine PLGA nanoparticles of DOX (DPNPs), so as to exploit the peculiarity of lymphatic vasculature underneath the subcutaneous layer of skin, which preferentially allows entry of only 10-100 nm sized particles. DPNPs were constructed using a novel solvent diffusion method aided by probe sonication, which resulted in an average size 95.43 ± 0.8 nm as per DLS, PDI 0.168 ± 0.03, zeta potential -7.38 ± 0.32, entrapment efficiency 75.58 ± 1.94%, and refrigerator stability of 7 days with respect to size in the optimized batch. TEM further substantiated the spherical shape of DPNPs along with their actual nonhydrated size as being well below 100 nm. FTIR analysis of DOX, dummy nanoparticles, and freeze-dried DPNPs revealed that the formulation step did not induce prominent changes in the chemical nature of DOX. The drug release was significantly altered (p < 0.05) with 64.6 ± 1.67% release in 48 h from DPNPs and was dictated by Fickian diffusion. Pharmacokinetic studies in Wistar rats further revealed that DPNPs caused a 16-fold prolongation in attainment of plasma Tmax and a 2-fold extension of elimination half-life (28.569 ± 1.27 h) at a dose of 5 mg/kg when compared to native drug (DOX solution) of the same strength. Contrastingly the trend was reversed in regional lymph nodes where Cmax for DPNPs (820 ± 84 ng/mg) was 4-fold greater, and lymphatic Tmax was attained in one-fourth of what was required for DOX solution. This size based preferential lymphatic targeting resulted in significantly greater in vivo antifilarial activity of DPNPs when compared to DOX solution as gauged by several parameters in Brugia malayi infected Mastomys coucha. Interestingly, the magnification in efficacy was obtained despite equivalent in vitro antifilarial activity of DOX solution and DPNPs against B. malayi worms.

Wolbachia endosymbiont of Brugia malayi elicits a T helper type 17-mediated pro-inflammatory immune response through Wolbachia surface protein.

Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B. malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4(+) T helper type 17 (Th17) cells and Th1 cytokines like interferon-γ and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor ß (TGF-ß) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4(+) Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-ß levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-ß secretion.

Homology modeling of NAD+-dependent DNA ligase of the Wolbachia endosymbiont of Brugia malayi and its drug target potential using dispiro-cycloalkanones.

Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD(+)-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD(+)-dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD(+) cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 µM), followed by compound 5 (IC50, 2.3 µM) and compound 1 (IC50, 2.9 µM). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

Moxidectin causes adult worm mortality of human lymphatic filarial parasite Brugia malayi in rodent models.

Moxidectin is a macrocyclic lactone belonging to milbemycin family closely related to ivermectin and is currently progressing towards Phase III clinical trial against human infection with the filaria Onchocerca volvulus (Leuckart, 1894). There is a single report on the microfilaricidal and embryostatic activity of moxidectin in case of the human lymphatic filarial parasite Brugia malayi (Brug, 1927) in Mastomys coucha (Smith) but without any adulticidal action. In the present study, the in vitro and in vivo antifilarial efficacy of moxidectin was evaluated on, B. malayi. In vitro moxidectin showed 100% reduction in adult female worm motility at 0.6 µM concentration within 7 days with 68% inhibition in the reduction of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye) (which is used to detect viability of worms). A 50% inhibitory concentration (IC50) of moxidectin for adult female parasite was 0.242 µM, for male worm 0.186 µM and for microfilaria IC50 was 0.813 µM. In adult B. malayi-transplanted primary screening model (Meriones unguiculatus Milne-Edwards), moxidectin at a single optimal dose of 20 mg/kg by oral and subcutaneous route was found effective on both adult parasites and microfilariae. In secondary screening (M coucha, subcutaneously inoculated with infective larvae), moxidectin at the same dose by subcutaneous route brought about death of 49% of adult worms besides causing sterilisation in 54% of the recovered live female worms. The treated animals exhibited a continuous and sustained reduction in peripheral blood microfilaraemia throughout the observation period of 90 days. The mechanism of action of moxidectin is suggested to be similar to avermectins. The in silico studies were also designed to explore the interaction of moxidectin with glutamate-gated chloride channels of B. malayi. The docking results revealed a close interaction of moxidectin with various GluCl ligand sites of B. malayi.

Molecular characterization of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi and antifilarial activity of specific inhibitors of the enzyme.

The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi, its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi. The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC50], 0.32 µM), followed by NSC-658343 (IC50, 4.13 µM) and NSC-657589 (IC50, 7.5 µM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors.

Structural modelling studies and immunoprophylactic potential of Brugia malayi DEAD Box RNA helicase.

DEAD Box RNA helicases are essential enzymes that are involved in RNA metabolic processes such as transcription, pre-mRNA splicing, translation initiation and RNA decay. We have previously over-expressed and biochemically characterized an immunodominant cDNA clone encoding DEAD box RNA helicase (BmL3-Helicase) isolated by immunoscreening of the larval stage cDNA library of Brugia malayi. In the current study, the 3D structure was determined and the immunoprophylactic efficacy of BmL3-Helicase was investigated by immunizing Mastomys coucha with the recombinant protein and subsequently challenging with B. malayi infective larvae. The immunization had an adverse outcome on the establishment of challenged larvae resulting in a 67.4% reduction in adult parasite recovery, a 86.7% decrease in the microfilarial density and profound sterility of the recovered female worms. The immune response thus generated was investigated by measuring the levels of specific antibodies including IgG subclasses, reactive oxygen species and cytokines.

Unresponsiveness of Mycobacterium w vaccine in managing acute and chronic Leishmania donovani infections in mouse and hamster.

The role of Mycobacterium w (Mw) vaccine as an immunomodulator and immunoprophylactant in the treatment of mycobacterial diseases (leprosy and pulmonary tuberculosis) is well established. The fact that it shares common antigens with leishmanial parasites prompted its assessment as an immunostimulant and as an adjunct to known anti-leishmanials that may help in stimulating the suppressed immune status of Leishmania donovani-infected individuals. The efficacy of Mw vaccine was assessed as an immunomodulator, prophylactically either alone or in combination with anti-leishmanial vaccine, as well as therapeutically as an adjunct to anti-leishmanial treatment in L. donovani-infected hamsters, representing a chronic human Visceral Leishmaniasis (VL) model. Similarly, its efficacy was also evaluated in L. donovani-infected BALB/c mice, representing an acute VL model. The preliminary studies revealed that Mw was ineffective as an immunostimulant and/or immunoprophylactant in hamsters infected with L. donovani, as estimated by T-cell immunological responses. However, in the BALB/c mice-VL model it appeared as an effective immunostimulant but a futile prophylactic agent. It is therefore inferred that, contrary to its role in managing tuberculosis and leprosy infections, Mw vaccine has not been successful in controlling VL infection, emphasizing the need to find detailed explanations for the failure of this vaccine against the disease.

Current drug targets for helminthic diseases.

More than 2 billion people are infected with helminth parasites across the globe. The burgeoning drug resistance against current anthelmintics in parasitic worms of humans and livestock requires urgent attention to tackle these recalcitrant worms. This review focuses on the advancements made in the area of helminth drug target discovery especially from the last few couple of decades. It highlights various approaches made in this field and enlists the potential drug targets currently being pursued to target economically important helminth species both from human as well as livestock to combat disease pathology of schistosomiasis, onchocerciasis, lymphatic filariasis, and other important macroparasitic diseases. Research in the helminths study is trending to identify potential and druggable targets through genomic, proteomic, biochemical, biophysical, in vitro experiments, and in vivo experiments in animal models. The availability of major helminths genome sequences and the subsequent availability of genome-scale functional datasets through in silico search and prioritization are expected to guide the experimental work necessary for target-based drug discovery. Organized and documented list of drug targets from various helminths of economic importance have been systematically covered in this review for further exploring their use and applications, which can give physicians and veterinarians effective drugs in hand to enable them control worm infections.

Improved antifilarial activity of ivermectin in chitosan-alginate nanoparticles against human lymphatic filarial parasite, Brugia malayi.

The current antifilarial treatments are not up to the mark partly due to deep location of filarial parasites in the human lymphatic system. We report here on the improvement in the antifilarial activity of ivermectin (IVM) using chitosan-alginate nanoparticles prepared by modified complex coacervation method. The nanoparticles were spherical having 155 nm size and 4.56 and 75.67% loading and entrapment efficiency respectively for IVM. The delivery system maintained the sustained release and significantly augmented the microfilaricidal (MIF) activity at a single low dose (200 µg/kg body weight, subcutaneously) in contrast to much higher dose of free ivermectin (400 µg/kg body weight, subcutaneously) against human lymphatic filariid, Brugia malayi in rodent host, Mastomys coucha. To substantiate increase in MIF activity, pharmacokinetics study was designed on Wistar rats which revealed a greater peak plasma concentration (45.3 ± 1.79 ng/ml), area under the concentration curve (298 ± 38.7 ng d/ml) and extended mean residence time (23.4 ± 8.56 days)of IVM in chitosan-alginate nanoparticles. Administration of 25 mg/kg of diethylcarbamazine following nanoparticle therapy significantly improved the MIF and macrofilaricidal action of encapsulated drug and was considered superior in this study.

Antifilarial activity of marine sponge Haliclona oculata against experimental Brugia malayi infection.

The present study incorporates the findings on in vitro and in vivo antifilarial activity in the marine sponge, Haliclona oculata using an experimental rodent infection of human lymphatic filarial parasite, Brugia malayi. The in vitro antifilarial action was determined on both adult female worms as well as microfilariae using two parameters viz. adverse effect on motility and inhibition in MTT reduction by the treated adult parasite over control worm. The antifilarial activity could be located in the methanol extract and one of its four fractions (chloroform). Bioactivity guided fractionation of chloroform fraction led to localization of in vitro activity in one of its eight chromatographic fractions. Methanol extract, chloroform fraction and one of the chromatographic fractions revealed IC(50) values of 5.00, 1.80, and 1.62µg/ml, respectively when adult B. malayi were exposed to these test samples for 72h at 37°C. Under similar exposure conditions, the IC(50) values for microfilariae were 1.88, 1.72 and 1.19µg/ml, respectively. The active test samples were found to be safe revealing >10 selectivity indices (SI) on the basis of cytotoxicity to Vero cells (monkey kidney cells) and therefore selected for in vivo evaluation against primary (adult B. malayi intraperitoneal transplanted jird) and secondary (subcutaneous infective larvae induced mastomys) screens. In primary jird model, the three test samples at 100mg/kg for five consecutive days by subcutaneous route demonstrated significant macrofilaricidal efficacy to the tune of 51.3%, 64% and 70.7% by methanol extract, chloroform and chromatographic fraction, respectively. The three samples demonstrated 45-50% macrofilaricidal activity with moderate embryostatic effect in secondary model at 5×500, 5×250 and 5×125mg/kg by oral route. Chromatographic fraction possessing highest antifilarial action was primarily found to be a mixture of four alkaloids Mimosamycin, Xestospongin-C, Xestospongin-D and Araguspongin-C in addition to few minor compounds.

Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host.

The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 µg/ml; mf = 13.17 µg/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 × 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 µg/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 µg/ml, CC50 = 212.5 µg/ml, SI = 889.1) and photogedunin (IC50 = 0.213 µg/ml, CC50 = 262.3 µg/ml, SI = 1231.4) at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to the presence of two pure compounds gedunin and photogedunin.

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite.

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.

In vitro and in vivo antifilarial potential of marine sponge, Haliclona exigua (Kirkpatrick), against human lymphatic filarial parasite Brugia malayi: antifilarial activity of H. exigua.

The present study reports on the antifilarial activity of a marine sponge Haliclona exigua (phylum Porifera). The crude methanol extract and n-butanol-soluble fraction killed adult Brugia malayi at 31.25-microg/ml concentration (both in motility and 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay) while the chloroform fraction was lethal at a lower concentration of 15.6 microg/ml. The activity could be located in a single molecule araguspongin C which brought about mortality of worm at 15.6 microg/ml. In vivo evaluation of the crude extract (5 x 500 mg/kg, orally) and the chloroform fraction (5 x 250 mg/kg, orally) in B. malayi-infected rodent host, Mastomys coucha, did not show any significant microfilaricidal actions; however, microfilarial densities in both the treated groups were significantly much lower than those of untreated group in contrast to standard filaricide diethylcarbamazine which exerted 79% microfilaricidal action on day 8 of treatment. Both these extracts also demonstrated adulticidal (macrofilaricidal) activity which was more pronounced in the chloroform fraction (50.2%). In addition, there was moderate adverse effect on the reproductive potential of female worms (crude extract 46.5%; chloroform 58.6%). The findings suggest that the marine sponge H. exigua possesses adulticidal and embryostatic action against human lymphatic filarial parasite B. malayi in experimental rodent model and this activity could be attributed to the presence of araguspongin C.

Antifilarial lead molecules isolated from Trachyspermum ammi.

Lymphatic filariasis is caused by infection with the parasitic filarial nematodes Wuchereria bancrofti, Brugia malayi and B. timori, transmitted by mosquitoes. The lack of an adulticidal drug poses a challenge to filariasis elimination, hence it is essential to develop an effective antifilarial drug which could either kill or permanently sterilize the adult worms. In the reported work the in vitro activity of a methanolic extract of fruits of Trachyspermum ammi (Apiaceae) against adult bovine filarial Setaria digitata worms has been investigated. A bioassay-guided fractionation was carried out by subjecting the crude extract to flash chromatography. HPLC analysis was done for the crude extract and active fraction. The crude extract and the active fraction showed significant activity against the adult S. digitata by both a worm motility and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assays. The isolated active principle was chemically characterized by IR, (1)H-NMR and MS analysis and identified as a phenolic monoterpene. It was screened for in vivo antifilarial activity against the human filarial worm B. malayi in Mastomys coucha, showing macrofilaricidal activity and female worm sterility in vivo against B. malayi. The findings thus provide a new lead for development of a macrofilaricidal drug from natural products.

RNA interference mediated knockdown of Brugia malayi UDP-Galactopyranose mutase severely affects parasite viability, embryogenesis and in vivo development of infective larvae.

BACKGROUND: Galactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp. This sugar maintains the integrity of parasite surface and is essential for virulence. UDP-Galactopyranose mutase (bmugm) plays a key role in Galf biosynthesis by catalyzing conversion of UDP-Galactopyranose into UDP-galactofuranose and knockout studies of the gene in Leishmania major, Mycobacterium and Aspergillus fumigatus displayed attenuated virulence while RNA interference study in C. elegans exhibited detrimental effects. Presence of UGM in several prokaryotic and eukaryotic microbial pathogens and its absence in higher eukaryotes renders it an attractive drug target. In the present study, RNA interference studies have been carried out to validate bmugm as an antifilarial drug target. METHODS: RNA interference studies using two different sequences of siRNAs targeting bmugm were carried out. The in vitro gene silencing of adult B. malayi parasites was undertaken to observe the effects on parasites. Infective larvae were also exposed to siRNAs and their in vivo development in jirds was observed. RESULTS: The in vitro gene silencing induced by siRNA1 and 2 individually as well as together knocked down the bmugm gene expression causing impaired viability of the exposed worms along with extremely reduced motility, abridged microfilarial release and adversely effected embryogenesis. The combinatorial in vitro gene silencing revealed marginally better results than both the siRNAs individually. Thus, infective larvae were treated with siRNA combination which showed downregulation of bmugm mRNA expression resulting into sluggish larval movements and/or death. The siRNA-treated actively motile larvae when inoculated intraperitoneally into jirds demonstrated highly reduced transformation of these larvae into adult worms with detrimental effects on embryogenesis. The effects of gene silencing were long-lasting as the adult worms developed from siRNA-treated larvae showed noticeable knockdown in the target gene expression. CONCLUSIONS: The validation studies undertaken here conclude that bmugm is essential for the proper development and survival of the parasite and support its candidature as an antifilarial drug target.