Lymphatic Filarial Parasite Research Articles

Immunomodulation of streptozotocin induced Type 1 diabetes mellitus in mouse model by Macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti

Helminth parasites modulate the host immune system to ensure a long-lasting asymptomatic form of infection generally, mediated by the secretion of immunomodulatory molecules and one such molecule is a homologue of human host cytokine, Macrophage migratory Inhibitory Factor (hMIF). In this study, we sought to understand the role of homologue of hMIF from the lymphatic filarial parasite, Wuchereria bancrofti (Wba-MIF2), in the immunomodulation of the Streptozotocin (STZ)-induced Type1 Diabetes Mellitus (T1DM) animal model. Full-length recombinant Wba-MIF2 was expressed and found to have both oxidoreductase and tautomerase activities. Wba-MIF2 recombinant protein was treated to STZ induced T1DM animals, and after 5 weeks pro-inflammatory (IL-1, IL-2, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and gene expressions were determined in sera samples and spleen respectively. Pro-inflammatory and anti-inflammatory cytokine levels were significantly (p<0.05) up-regulated and down-regulated respectively, in the STZ-T1DM animals, as compared to treated groups. Histopathology showed macrophage infiltration and greater damage of islets of beta cells in the pancreatic tissue of STZ-T1DM animals, than Wba-MIF2 treated STZ-T1DM animals. The present study clearly showed the potential of Wba-MIF2 as an immunomodulatory molecule, which could modulate the host immune system in the STZ-T1DM mice model from a pro-inflammatory to anti-inflammatory milieu.

Anti-filarial efficacy of Centratherum anthelminticum: unravelling the underlying mechanisms through biochemical, HRAMS proteomics and MD simulation approaches.

Traditionally, Centratherum anthelminticum (CA) has been reported to be a potent anti-filarial, however no reports are available detailing its mechanism of action against filarial parasites. In this study, we have evaluated the anti-filarial activity of CA against lymphatic filarial parasites Setaria cervi using ex vivo biochemical, proteomics and in silico approaches. The motility and viability of the parasites decreased significantly after treatment with CA concentrations of ≥125 μg mL-1. An increase in lipid peroxidation (51.92%), protein carbonylation (48.99%), NADPH oxidase (88.88%) activity and decrease in the glutathione (GSH) (-39.23%), glutathione reductase (GR) (-60.17%), and glutathione S-transferase (GST) (-50.48%) activity was also observed after CA treatment. The proteomics analysis was performed by two-dimensional gel electrophoresis and high-resolution accurate mass spectrometry (HRAMS). In total, 185 proteins were differentially expressed (DEPs) following CA treatment. The major DEPs were mostly involved in tRNA processing, biosynthetic processes, metabolic activities, protein transport, the tricarboxylic acid cycle, protein translation, and stress response. The UPLC-ESI-MS/MS analysis of CA extract revealed the presence of 40 bioactive compounds. Further the docking analysis showed 10 CA bioactive compounds to have high binding affinity towards antioxidant proteins of filarial parasites. Additionally, MD simulation studies showed stable interactions (RMSF ≤ 10 Å) of 3-O-methylquercitin, quinic acid, gentisic acid, and vanillin with filarial antioxidant enzymes/proteins. To our knowledge, this is the first report detailing the molecular mechanism of anti-filarial activity of CA, which can be further evaluated for the development of new anti-filarial formulations.

Deciphering the anti-filarial potential of bioactive compounds from Ocimum sanctum: a combined experimental and computational study

Context The anthelminthic effect of Ocimum species (Lamiaceae) has been reported, however, its anti-filarial effect has not been explored to date. Objective This study evaluates the effect of Ocimum sanctum L. (OS) against lymphatic filarial parasites. Material and methods The ethanol extract of OS (EOS) leaves was tested for anti-filarial activity against Setaria cervi. Equal size and number (n = 10) of adult female S. cervi worms were incubated in 125, 250 or 375 μg/mL EOS extract for 6 h at 37 °C. The OS bioactive components were identified by UPLC-ESI-MS/MS and subjected to docking and molecular dynamics (MD) simulation against filarial antioxidant proteins. Results The EOS significantly inhibited the motility of adult female S. cervi after 6 h of incubation. The motility was found to be reduced by 53.7% in 375 µg/mL and 43.8% in 250 µg/mL EOS after 6 h of treatment. The UPLC-ESI-MS/MS analysis of ethanol extract of O. sanctum revealed the presence of 13 bioactive compounds. The docking analysis showed eight OS bioactive compounds to have high binding affinity (> 4.8 kcal/mol) towards antioxidant proteins of filarial parasites. Additionally, MD simulation studies showed significant impact of (RMSD ≤ 10 Å) chlorogenic acid, luteolin and ursolic acid on filarial antioxidant enzymes/proteins. To our knowledge, this is the first report of the anti-filarial activity of Ocimum sanctum. Discussion and conclusions The effect of EOS and OS bioactive components on human filarial parasites can be further evaluated for the development of new anti-filarial formulations.

Identification of promising nutraceuticals against filarial immune-modulatory proteins: insights from in silico and ex vivo studies.

Lymphatic filariasis is a neglected tropical disease affecting over 863 million people in 47 countries of the world. The anti-filarial drugs, diethylcarbamazine, albendazole, and ivermectin, are effective only at the larval stages and have proven completely ineffective as adulticides. Besides this, a long-term use of these drugs is associated with several side effects including drug toxicity. Nutraceuticals have emerged as better alternatives for long term treatments due to their safety and lesser side effects. In the present work, we have used drug docking analysis and molecular dynamics simulation approaches to explore the effect of anti-inflammatory nutraceuticals against the immune-modulatory proteins of filarial worms. The filarial proteins enolase, ES-62 precursor, serpin, and cystatin, which are highly efficient in host immune modulation were targeted with more than 50 nutraceuticals. In the in silico study nutraceuticals such as naringin, β-carotene, and emodin showed higher binding efficacy and lower dissociation constant as compared to anti-filarial drugs. Molecular dynamics simulation results showed that immune-modulatory proteins formed highly stable complexes with naringin, β-carotene, and emodin over the entire MD simulation run. The nutraceutical emodin formed the most stable system in silico and hence its effect was investigated on adult filarial parasites under ex vivo conditions too. Emodin significantly affected the motility, viability, ROS production, and genomic DNA fragmentation of filarial parasites. Further in vivo and in vitro studies will help in understanding the mechanism of action of emodin at the molecular level and would help in the development of more effective anti-filarial drugs.

Mitochondrial Genome of Brugia malayi Microfilariae Isolated From a Clinical Sample

Lymphatic filariasis is a neglected parasitic disease that is a leading cause of long-term disability. Information obtained from genome sequencing of filarial worm can help us identify systems in the worm that are likely to be useful for novel drug design. Brugia (B.) malayi is still the only lymphatic-dwelling filarial parasite with a nearly complete, fully annotated, and published genome. However, most previous studies were based on the FR3 strain of B. malayi, which originally was isolated from a human patient, and was adapted to the rodent model, then maintained in laboratories for more than 60 years. It is uncertain whether genetic variation exists, thus, sequencing of clinical isolates of lymphatic dwelling filarial parasites is a high priority. Here, we report for the first time the complete mitochondrial genome of B. malayi microfilariae from clinical isolate. Complete mitochondrial (mt) genome of the microfilariae isolated from a blood sample taken from a Thai subject living in Narathiwat Province, which is an endemic area of brugian filariasis, was assembled with sequencing reads obtained by Illumina sequencing. Gene annotation, phylogenetic analysis and single nucleotide polymorphism (SNP) were deployed. A complete 13,658-bp mt genome of B. malayi microfilaria was obtained, and it shows 68x coverage. Based on gene annotation, the mt genome consists of 12 protein-coding, two rRNA, and 23 tRNA genes. Phylogenetic analysis using all protein sequences of DNA sequences of mt genome or cytochrome c oxidase subunit I (COX1) revealed a close relationship among three lymphatic filariae (i.e., B. timori, zoonotic B. pahangi, and Wuchereria spp.). The SNPs in the COX1 gene can differentiate microfilariae of B. malayi in human from those found in canine. Furthermore, the number, order and transcription, and direction of B. malayi microfilariae mitochondrial genes were the same as those found in the FR3 strain of B. malayi. The comparison on mitochondrial genome of B. malayi could have important implications on the development of a new intervention or vaccine to treat or prevent this disease in endemic areas/regions around the world.

Role of Glutathione in Chalcone Derivative Induced Apoptosis of Brugia malayi and its Possible Therapeutic Implication.

Oxidative stress is an essential component of innate response against microbes. The oxidative impact has a very subtle connection with apoptosis. Our previous work indicated presumptive evidence of apoptosis by the chalcone derivatives against the human lymphatic filarial parasite. Evidence suggests the involvement of glutathione-S-transferase (GST) in the mechanism of action of chalcone drugs. In the present study, we explored the implications of redox status in apoptosis of the parasite by this drug. Treatment with the representative drug, 4t, significantly decreased GSH level and increased GST activity in the Brugia malayi microfilariae (Mf) in comparison to Mf without 4t treatment. Drug-induced loss of motility of the parasites was reversed by the treatment with GSH (41%) and NAC (19%). A significant fall in rGST activity was observed due to drug addition, which could be reversed by the addition of GSH co-substrate, but not with the re-addition of rGST, indicating a vital role of GSH. In silico study demonstrated a favorable drug-GST enzyme interaction. Oxidative stress was reflected by increased protein carbonylation and intracellular reactive oxygen species level, in the drug-treated parasite. Mitochondrial oxygen consumption was reduced by the drug, which was reversed on the addition of GSH. Mitochondrial dysfunction was confirmed by MTT and cytochrome c assay. Apoptosis was confirmed by the inhibition in PARP activity. We conclude that the depletion of GSH by chalcone with concomitant mitochondrial dysfunction revealed a novel rationale of apoptosis in the parasite. Such a mechanism might have wide therapeutic implications.

Fecundity of adult female worms were affected when Brugia malayi infected Mongolian gerbils were immunized with a multivalent vaccine (rBmHAXT) against human lymphatic filarial parasite

A multivalent recombinant fusion protein prophylactic vaccine, rBmHAXT developed against lymphatic filariasis (LF) demonstrated over 57% protection against challenge infection in rhesus macaque model. Currently, we do not know if the rBmHAXT vaccination has any effect on adult worms and/or on the fecundity of adult female worms. Thus, the major focus of this study was to determine the effect of rBmHAXT vaccination on Brugia malayi infected mongolian gerbils. We performed two sets of experiments. In the first set of experiment, gerbils were infected with 100 B. malayi L3. After confirming the establishment of infection, four rounds of DEC treatment and rBmHAXT vaccination was given. Results showed that following vaccination with rBmHAXT, the microfilaria (Mf) count was significantly decreased in all vaccinated animals compared to controls. At the end of these experiments, we collected and counted the established adult worms. There was a 36% reduction in the recovery of adult female worms, which might account for the low Mf load in vaccinated animals. In the second set of experiments, animals were vaccinated first with rBmHAXT followed by surgically implanting adult male or female B. malayi parasites into the peritoneal cavity to determine the effect of vaccination on each sex of the parasite. Our results show that the rBmHAXT vaccination has no effect on male adult worms compared to controls. However, there was 40% reduction in the Mf load in vaccinated animals that were transplanted with adult female worms. These findings suggested that the rBmHAXT vaccination has potential damaging effect on the fecundity of adult female worms. Scanning electron microscopy studies showed cuticular damage on the surface of adult female worms. These studies thus show that the rBmHAXT vaccination in infected gerbils has partial microfilaricidal effect and potentially affect the fecundity of adult female worms.

Structural, molecular, functional and immunological characterization of Wuchereria bancrofti-galectin

Galectins regulate growth and differentiation of immune cells and inflammation through their carbohydrate-binding function in humans, while also play a role in parasite survival. This study focused on the galectin of lymphatic filarial parasite Wuchereria bancrofti (Wb-Gal). The multiple sequence alignment with other galectins showed that the Wb-Gal belonged to galactoside binding lectin family, particularly tandem repeat type galectin-9. A homology model of Wb-Gal was developed in the I-TASser server using high similarity 3D structures with a quality score of 89.5. Molecular docking and dynamics studies revealed that the CCRD and NCRD of Wb-Gal bind with galactose and lactose. Further, Wb-Gal was cloned into the pET28 vector, expressed in E. coli Rosetta strain and purified by affinity chromatography. In the hemagglutination assays, the rWb-Gal bound to lactose, galactose, and glucose. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) using different clinical filarial sera showed that the IgG and IgM response was against Wb-Gal x very high in all filarial clinical groups, whereas the IgA and IgG2 response was minimum to negligible. There was an enhanced response of IgG1 and IgG4 antibodies in Microfilaremics (MF) cases compared to Chronic Pathology (CP) and Endemic Normal (EN) individuals. Interestingly, the IgE response was comparatively higher in EN than MF and CP. These studies show that Wb-Gal is a member of the lectin family of proteins binding to different carbohydrates and may have an important role in the pathophysiology of filarial infection which needs to be investigated in greater detail.

In vitro apoptotic effect on human lymphatic filarial parasite by piperidine derivatives and thymidine reversal study.

A novel library of synthetic piperidine derivatives was used to screen against human lymphatic filarial parasite Brugia malayi. Piperidine has earlier been reported to have effect against parasites including rodent filarial nematodes. Compounds with hydroxyl substitutions (4Q and 4H) showed marked antifilarial effect. Molecular docking of 4H derivative showed more favorable thermodynamic parameters against thymidylate synthase of B. malayi than human counterpart. A wide difference between IC50 and LD50 ensured the therapeutic safety of the candidates against the filarial parasites. Addition of thymidine to the treatment regimen led to a significant reversal of antifilarial effect of 4H that confirmed inhibition of thymidylate synthase as pharmacological rationale. Apoptosis induced in the parasite as a consequence of probable inhibition of thymidylate synthase was studied by acridine orange/ethidium bromide fluorescent staining and poly (ADP-ribose) polymerase activity inhibition. Involvement of mitochondria was confirmed by decreased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) conversion and increased cytosolic cytochrome c level in 4H treated microfilariae, compared with the untreated microfilariae. Moreover, Michael adduct of chalcone targeting dihydrofolate reductase and piperidine targeting thymidylate synthase demonstrated synergistic effect on the parasite, indicating the importance of inhibition of DNA synthesis by combined effect. In conclusion, piperidine derivatives with hydroxyl substitution have a great therapeutic potential with an apoptotic rationale involving mitochondrial pathway, due to possible inhibition of parasitic thymidylate synthase.

Polyphenols-Induced Apoptosis by Murraya Koenigii against Lymphatic Filarial Parasite and Its Therapeutic Implication

ABSTRACTThe present study was designed to explore and validate the antifilarial activity of Murraya koenigii leaf extracts against Brugia malayi parasite. Anti-microfilarial activity of M. koenigii leaves extracted in methanol and ethanol (along with polyphenols depleted fractions) was determined. Ethanolic extract showed higher polyphenols content and anti-microfilarial activity compared to methanolic extract. Polyphenols depletion reduced such activity of ethanolic but not of methanolic extract. Ethanolic extract had lower alkaloids than methanolic extract. Ethanolic extract of M. koenigii leaves caused oxidative stress with concomitant apoptosis along with mitochondrial damage.Abbreviations: DEC: Diethyl carbamazine; DNPH: 2, 4 dinitro phenyl hydrazine reagent; DTNB: 5,5-dithio-bis-(2-nitrobenzoic acid); EB/AO: Ethidium bromide/Acridine orange; MDA: Mass Drug Administration; Mf: Microfilariae; PBS: Phosphate buffered saline; TCA: Trichloroacetic acid; WHO: World Health Organization

The Genetic Polymorphisms of 24 Base Pair Duplication and Point G102S of Human Chitotriosidase to Bancroftian Filariasis at the Thai⁻Myanmar Border.

Lymphatic filariasis, caused by lymphatic filarial parasites, Wuchereria bancrofti, and Brugia malayi, causes significant morbidity and disability to 120 million people in the tropics and subtropics. Chitin has an important role for embryogenesis in adult worms and is a component of microfilaria sheath. Human chitotriosidase (CHIT1) is a chitin-degrading enzyme which provides a protective role against chitin-containing pathogens. Here, we determined the association of CHIT1 polymorphisms with susceptibility to bancroftian filariasis (BF) in 88 individuals at the Thai–Myanmar border. Two common polymorphisms of CHIT1, contributing inactive CHIT protein, including 24 base pair (24 bp) duplication in exon 10, and p. G102S in exon 4 were genotyped by allele-specific Polymerase Chain Reaction (PCR) and PCR sequencing, respectively. Unexpectedly, genotype frequencies of 24 bp duplication insertion homozygous (INS/INS) were significantly higher in endemic normal (EN) (40.0%) than BF patients (31.4%). In contrast, genotype frequencies of p. G102S homozygous (A/A) in BF patients (21.6%) was higher than in EN (19.0%) without statistical difference. Mutant allele frequencies of 24 bp duplication were 0.6125 (98/160) and p. G102S were 0.392 (69/176). Genotype and allele frequencies of CHIT1, 24 bp duplication, and p. G102S, showed no association with BF patients.

Synthesis, Structure Elucidation, Homology Modeling and Antifilarial Activity of 7‐Benzamidocoumarin Derivatives

AbstractA series of 7‐benzamidocoumarin derivatives 10–25 starting from 7‐amino coumarins 7 and 8 has been synthesized, characterized and evaluated in vitro for antifilarial activity against the human lymphatic filarial parasite, Brugia malayi. Compounds 13 and 20–23 showed permanent paralysis of the worm with 90–95% inhibition of motility of adult worms at 10 μM. All the synthesized compounds were docked on the modeled receptor of Onchocerca volvulus chitinase OvCHT1. Compound 13 with binding energy of −7.95 Kcal/mol showing three hydrogen bonds with the active site of the enzyme emerged as the best inhibitor.

Mosquitoes and the Lymphatic Filarial Parasites: Research Trends and Budding Roadmaps to Future Disease Eradication.

The mosquito-borne lymphatic filariasis (LF) is a parasitic, neglected tropical disease that imposes an unbearable human scourge. Despite the unprecedented efforts in mass drug administration (MDA) and morbidity management, achieving the global LF elimination slated for the year 2020 has been thwarted by limited MDA coverage and ineffectiveness in the chemotherapeutic intervention. Moreover, successful and sustainable elimination of mosquito-vectored diseases is often encumbered by reintroduction and resurgence emanating from human residual or new infections being widely disseminated by the vectors even when chemotherapy proves effective, but especially in the absence of effective vaccines. This created impetus for strengthening the current defective mosquito control approach, and profound research in vector–pathogen systems and vector biology has been pushing the boundaries of ideas towards developing refined vector-harnessed control strategies. Eventual implementation of these emerging concepts will offer a synergistic approach that will not only accelerate LF elimination, but also augurs well for its future eradication. This brief review focuses on advances in mosquito–filaria research and considers the emerging prospects for future eradication of LF.

Molecular cloning and characterization of protein disulfide isomerase of Brugia malayi, a human lymphatic filarial parasite.

Lymphatic filariasis results in an altered lymphatic system and the abnormal enlargement of body parts, causing pain, serious disability and social stigma. Effective vaccines are still not available nowadays, drugs against the disease is required. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum which is involved in folding and chaperone activities in different biological systems. Here, we report the enzymatic characterization of a Brugia malayi Protein disulfide isomerase (BmPDI), which was expressed and purified from Escherichia coli BL21 (DE3). Western blotting analysis showed the recombinant BmPDI could be recognized by anti-BmPDI Rabbit serum. The rBmPDI exhibited an optimum activity at pH 8 and 40 °C. The enzyme was inhibited by aurin and PDI inhibitor. Recombinant BmPDI showed interaction with recombinant Brugia malayi calreticulin (rBmCRT). The three-dimensional model for BmPDI and BmCRT was generated by homology modelling. A total of 25 hydrogen bonds were found to be formed between two interfaces. There are 259 non-bonded contacts present in the BmPDI-BmCRT complex and 12 salt bridges were formed in the interaction.

Functional Impairment of Murine Dendritic Cell Subsets following Infection with Infective Larval Stage 3 of Brugia malayi.

Filarial parasites cause functional impairment of host dendritic cells (DCs). However, the effects of early infection on individual DC subsets are not known. In this study, we infected BALB/c mice with infective stage 3 larvae of the lymphatic filarial parasite Brugia malayi (Bm-L3) and studied the effect on fluorescence-activated cell sorter (FACS)-sorted DC subsets. While myeloid DCs (mDCs) accumulated by day 3 postinfection (p.i.), lymphoid DCs (LDCs) and CD8+ plasmacytoid DCs (pDCs) peaked at day 7 p.i. in the spleens and mesenteric lymph nodes (mLNs) of infected mice. Increased tumor necrosis factor alpha (TNF-α) but reduced interleukin 12 (IL-12) and Toll-like receptor 4 (TLR4), -6, and -9 and reciprocal secretion of IL-4 and IL-10 were also observed across all DC subsets. Interestingly, Bm-L3 increased the expression of CD80 and CD86 across all DC subsets but decreased that of major histocompatibility complex class II (MHC-II) on mDCs and pDCs, resulting in their impaired antigen uptake and presentation capacities, but maximally attenuated the T-cell proliferation capacity of only mDCs. Furthermore, Bm-L3 increased phosphorylated p38 (p-p38), but not p-ERK, in mDCs and LDCs but downregulated them in pDCs, along with differential modulation of protein tyrosine phosphatases SHP-1, TCPTP, PTEN, and PTP1B across all DC subsets. Taken together, we report hitherto undocumented effects of early Bm-L3 infection on purified host DC subsets that lead to their functional impairment and attenuated host T-cell response.

Sulfonamide chalcones: Synthesis and in vitro exploration for therapeutic potential against Brugia malayi

Keeping in mind the immense biological potential of chalcones and sulfonamide scaffolds, a library of sulfonamide chalcones has been synthesized and evaluated for in vitro antifilarial assay against human lymphatic filarial parasite Brugia malayi. Experimental evidence showcased for the first time the potential of some sulfonamide chalcones as effective and safe antifilarial lead molecules against human lymphatic filarial parasite B. malayi. Sulfonamide chalcones 4d, 4p, 4q, 4t and 4aa displayed the significantly wide therapeutic window. Particularly chalcones with halogen substitution in aromatic ring proved to be potent antifilarial agents against Brugia malayi. Sulphonamide chalcones with lipophilic methyl moiety (4q and 4aa) at para position of terminal phenyl rings of compounds were found to have remarkable antifilarial activities with therapeutic efficacy. Observed preliminary evidence of apoptosis by effective chalcone derivatives envisaged its fair possibility to inhibit folate pathway with consequent defect in DNA synthesis.

Antifilarial Activity of Eucalyptus globulus Labill. Leaves Against Brugia malayi

The present study is aimed to evaluate the antifilarial activity of Eucalyptus globules Labill. (Myrtaceae) against human lymphatic filarial parasite Brugia malayi in vitro and in vivo. The ethanolic extract of the leaves was tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active sample was 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 ethanolic extract of the leaves of the E. globulus was tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active sample was further evaluated in vivo in B. malayi. The ethanolic extract was active in vitro (IC50: adult = 62.5 ?g/ml; mf = 31.2. ?g/ml) where it demonstrated 66.7% adulticidal and embryostatic effect on B. malayi in Mastomys at a dose of 5 × 100 mg/kg by oral route. The antifilarial test conducted was at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 66.7% transplanted adult B. malayi in the peritoneal cavity of jirds in addition to noticeable microfilaricidal action on the day of autopsy. The findings revealed that the extract from the leaves of E. globulus contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which may be further explored to new antifilarial agents.Bangladesh Pharmaceutical Journal 19(1): 44-47, 2016

Filarial Abundant Larval Transcript Protein ALT-2: An Immunomodulatory Therapeutic Agent for Type 1 Diabetes.

Type 1 diabetes (T1D) that accounts for about 5-10% of all diabetes cases results from the autoimmune destruction of the insulin-producing beta cells in the pancreas. It is characterized by severe inflammatory reaction mediated by pronounced T helper type-1 response. Parasitic infections having the ability to skew the host immune responses towards type-2 type as a part of their defense mechanism are able to induce protection against autoimmune diseases like T1D. Hence, the present study is undertaken to explore a recombinant abundant larval transcript protein of the human lymphatic filarial parasite Brugia malayi (rBmALT-2), a known anti-inflammatory molecule for its therapeutic effect on streptozotocin (STZ)-induced T1D in mice. The diabetic mice on treatment with rBmALT-2 showed a significant (p<0.0005) decrease in their fasting blood glucose levels. By the end of the second week after the initiation of treatment with the rBmALT-2, 28% of the diabetic mice became normal and none of them were diabetic by the end of 5th week. The anti-diabetic effect of rBmALT-2 significantly correlated with the concomitant redressal of the pancreatic histopathological damage caused by STZ assault (rho=0.87; p<0.0005). The sera of rBmALT-2 treated diabetic mice had increased levels of IgG1 antibodies associated with decreased IgG2a antibodies against the principal autoantigen insulin. The splenocyte proliferative response and the cytokine release in the treated mice showed marked bias against inflammation skewing the immune response to Th-2 type. From this study, it can be envisaged that that filarial proteins like rBmALT-2 with effective immunomodulatory activity and anti-diabetic effect are promising alternative therapeutic agents for T1D.

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.

Brugia malayi Asparaginyl - tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis

A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis.