Shailja Misra Bhattacharya

Bacterially expressed recombinant envelope protein domain III of Japanese encephalitis virus (rJEV-DIII) elicits Th1 type of immune response in BALB/c mice

Japanese encephalitis is a major cause of encephalitis in Asia. Cases occur largely in rural areas of the South and East Asian region resulting in significant morbidity and mortality. Multiple vaccines exist to control Japanese encephalitis, but all suffer from problems. Envelope protein domain III of Japanese encephalitis virus is involved in binding to host receptors and it contains specific epitopes that elicit virus-neutralizing antibodies. Earlier, the protective efficacy of domain III has been evaluated in mice by some researchers, but these studies are lacking in explanation of humoral and cellular immune responses. We have earlier reported cloning, expression, purification and in vitro refolding of Japanese encephalitis virus envelope protein domain III (rJEV-DIII). Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80. In the present study, we have evaluated the immunomodulatory potential of refolded rJEV-DIII protein in BALB/c mice with Freunds complete/incomplete adjuvants. Mice were tested for humoral immune response by ELISA. Cell-mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. The rJEV-DIII generated high IgG antibody and its isotypes (IgG2a and IgG3) and induced significant expression of INF-gamma and IL-2 cytokines. The rJEV-DIII induced significant lymphoproliferation of splenocytes. In conclusion rJEV-DIII induced Th1 type of immune response which plays an important role in protection for intracellular pathogens.

In vitro silencing of Brugia malayi trehalose-6-phosphate phosphatase impairs embryogenesis and in vivo development of infective larvae in jirds.

Background The trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP) is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes. Methodology and Principal Findings In the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds. Conclusions/Significance The present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.

Nanopharmaceuticals to target antifilarials: a comprehensive review

Introduction: Currently emphasized conventional chemotherapies for the elimination of lymphatic filariasis (LF) are imperfect due to unfocused targeting of poorly water-soluble antifilarial drugs. The profound location of drug targets (filarial parasites or wolbachia) within the complex anatomy of lymphatic tissues often necessitates prolonged treatment schedules with high doses leading to undesired side effects and poor patient compliance. Therefore, we need to reformulate antifilarial drugs taking the advantages of nanotechnology through a wide range of nanomedical carriers, which improve drug efficacy, increase bioavailability, and diminish toxicity. Areas covered: Connotations of drug delivery systems (DDSs) to target lymphatic filaroids or wolbachia and systemic microfilaria have been discussed. The potentials of liposomes and solid lipid nanoparticles for the treatment of LF are highlighted. Various critical factors, viz optimal size range, surface properties, preferred pH, mechanism of reticuloendothelial avoidance, and control of the release of antifilarial agents for safe elimination of parasites, are enclosed to design a novel DDS for LF. The review of nanotechnological approaches to improve antifilarial chemotherapy will help to resolve existing technological gaps. Expert opinion: Precincts in the antifilarial discovery programs can never be overcome by conventional methods. Nanomedicine encompasses wide-range solution for each single problem (i.e., from poor solubility to nonspecific targeting of antifilarial agents) for the cure of LF at low costs and may reduce the economic burden of LF diseases. Advances in nanotechnology loom will certainly come forward as silver bullets in the near future for quick diagnosis, control, and elimination of this tropically neglected disease.

Chitosan coated alginate micro particles for the oral delivery of antifilarial drugs and combinations for intervention in Brugia malayi induced lymphatic filariasis

In the present study chitosan (Chi) coated alginate micro particles (MPs) were prepared by a spray drying technique for the effective delivery to lymphatics via combination therapy. The optimized MPs were spherical of less than 5 μm in size with loading and entrapment efficiencies of 14.13 ± 1.1% and 41.28 ± 1.5% for diethylcarbamazine (DEC), and 14.93 ± 0.58% and 43.51 ± 1.9% for doxycycline (DOX) respectively. The physical mixture and drug entrapped MPs showed good stability and no interaction was evident from FT-IR, XRD and thermal analysis. In vitro release studies revealed that the drug release pattern was sustained beyond 16 h in simulated gastro-intestinal fluids for DEC and DOX from the MPs. In vivo ileal uptake and histopathological studies in rats divulged the uptake of the MPs by intestinal epithelium. Further in vitro antifilarial activity studies demonstrated notable killing by the combination therapy compared to individual treatments with Chi–DOX MPs and Chi–DEC MPs. A combined dose of 25 mg kg−1 and 10 mg kg−1 in a 2.5 : 1 ratio of DEC and DOX respectively, following the oral MP therapy, significantly improved the microfilaricidal and macrofilaricidal action of the in vitro estimations. In vivo experiments of infected jirds (Meriones unguiculatus) were carried out. The macrofilaricidal/adulticidal action was enhanced by 2 fold with Chi–DOX MPs in combination with Chi–DEC MPs, microfilaricidal enhancements of the Chi–DEC & Chi–DOX combination are also inferred.

Antifilarial Activity of Gymnema SylvestreR. Br Leaves Against Brugia Malayi

The present study is aimed to evaluate the anti-filarial activity of Gymnema sylvestre (Asclepiadaceae), 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 G. sylvestre 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= 65.0 μg/ml; mf = 32.5. μg/ml) where it demonstrated 65.0% adulticidal and embryostatic effect on B malayi in Mastomys at a dose of 5 × 100mg/kg by oral route. The antifilarial test conducted was at 5×100mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 65.0% 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 G. Sylvestre 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.

Circulating filarial antigen detection in brugian filariasis

Human lymphatic filariasis (LF) is a major cause of disability globally. The success of global elimination programmes for LF depends upon effectiveness of tools for diagnosis and treatment. In this study on stage-specific antigen detection in brugian filariasis, L3, adult worm (AW) and microfilarial antigenaemia were detected in around 90-95% of microfilariae carriers (MF group), 50-70% of adenolymphangitis (ADL) patients, 10-25% of chronic pathology (CP) patients and 10-15% of endemic normal (EN) controls. The sensitivity of the circulating filarial antigen (CFA) detection in serum samples from MF group was up to 95%. In sera from ADL patients, unexpectedly, less antigen reactivity was observed. In CP group all the CFA positive individuals were from CP grade I and II only and none from grade III or IV, suggesting that with chronicity the AWs lose fecundity and start to disintegrate and die. Amongst EN subject, 10-15% had CFA indicating that few of them harbour filarial AWs, thus they might not be truly immune as has been conventionally believed. The specificity for antigen detection was 100% when tested with sera from various other protozoan and non-filarial helminthic infections.