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.

Therapeutic efficacy of poly (lactic-co-glycolic acid) nanoparticles encapsulated ivermectin (nano-ivermectin) against brugian filariasis in experimental rodent model.

The present study reports on the antifilarial activity of poly (lactic-co-glycolic acid) nanoparticles encapsulated ivermectin (nano-IVM) against human lymphatic filariid Brugia malayi in rodent host Mastomys coucha. Nano-IVM was prepared and optimized by nanoprecipitation method. The selected nano-IVM (F5) showed a uniform spherical shape with 96 nm diameter and 74.12 % entrapment efficiency, and when used at a suboptimal dose of 100 µg/kg body weight, completely eliminated filarial parasites from systemic circulation on 60 days post-infection in animals inflicted with B. malayi. In contrast, the coadministration of nano-IVM (F5) along with standard filaricide diethylcarbamazine (DEC) was found to be competent enough to suppress microfilarial stage of parasites and successfully eliminated microfilaria at 45 days posttreatment. However, the free form of both the drugs alone or in combination was unable to impart such suppression and followed by recurrence of the infection. Interestingly, nano-IVM (F5) was also found to be effective against adult stage parasites causing 36.67 % worm mortality and 75.89 % in combination with DEC; however, female sterilization remain almost similar. Thus, the combination of entrapped IVM with DEC exhibited enhanced microfilaricidal and marginally better macrofilaricidal efficacy than any of the single formulation or drugs combination.

Stage-specific antibody response against two larval stages of Brugia malayi in different clinical spectra of brugian filariasis.

CONTEXT: T-cell hypo-responsiveness in microfilaria (Mf) carriers against the microfilarial stage antigen of Brugia malayi has been described, but no study has been carried out to assess antibody dynamics against stage-specific antigens. AIM: The work was carried out with the aim to assess stage-specific antibody responses against L3 and microfilarial stage antigens in brugian filariasis in an endemic area. SETTING AND DESIGN: Patients with different clinical spectra of brugian filariasis were recruited to evaluate antibody responses to brugian antigens. SUBJECTS AND METHODS: Serum samples were collected from patients with different clinical spectra and antibody response was evaluated for total immunoglobulin G (IgG), IgG isotypes (IgG1, IgG2, IgG3, IgG4) and immunoglobulin E (IgE) response to L3 and microfilarial stage by enzyme-linked immunosorbent assay. STATISTICAL ANALYSIS: Paired t-test and one-way analysis of variance were carried out to analyze the data. RESULTS: L3 and microfilarial stage antigens showed almost similar antibody responses in adenolymphangitis (ADL) and chronic pathology (CP) patients, however, diminished antibody response was observed with Mf stage antigen, especially with microfilaraemia. ADL patients had minimum antibody levels of all isotypes except IgG2 on day 0 which showed an increase subsequently, indicating suppression of antibody response during filarial fever. CP patients showed increase in IgE and decrease in IgG4 antibodies on day 365 indicating that these differences may be due to recent conversion into CP. CONCLUSION: A prominent hyporesponsiveness in microfilaraemic individuals against microfilarial stage, but not against the L3 stage of the same parasite was observed, concluding stage-specificity in humoral immune response in brugian filariasis.

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.

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.

The n-hexane and chloroform fractions of Piper betle L. trigger different arms of immune responses in BALB/c mice and exhibit antifilarial activity against human lymphatic filarid Brugia malayi.

Modulation of immune functions by using herbal plants and their products has become fundamental regime of therapeutic approach. Piper betle Linn. (Piperaceae) is a widely distributed plant in the tropical and subtropical regions of the world and has been attributed as traditional herbal remedy for many diseases. We have recently reported the antifilarial and antileishmanial efficacy in the leaf extract of Bangla Mahoba landrace of P. betle which is a female plant. The present report describes the in vivo immunomodulatory efficacy of the crude methanolic extract and its n-hexane, chloroform, n-butanol fractions of the female plant at various dose levels ranging between 0.3 and 500 mg/kg in BALB/c. Attempts were also made to observe antifilarial activity of the active extracts and correlate it with the antigen specific immune responses in another rodent Mastomys coucha infected with human lymphatic filarial parasite Brugia malayi. The crude methanol extract and n-hexane fraction were found to potentiate significant (p<0.001) enhancement of both humoral (plaque forming cells, hemagglutination titre) as well as cell-mediated (lymphoproliferation, macrophage activation, delayed type hypersensitivity) immune responses in mice. The flow cytometric analysis of splenocytes of treated mice indicated enhanced population of T-cells (CD4(+), CD8(+)) and B-cells (CD19(+)). The n-hexane fraction (3 mg/kg) was found to induce biased type 2 cytokine response as revealed by increased IL-4(+) and decreased IFN-gamma(+) T-cell population while the chloroform fraction (10 mg/kg) produced a predominant type 1 cytokines. Crude methanolic extract (100 mg/kg) demonstrated a mixed type 1 and type 2 cytokine responses thus suggesting a remarkable immunomodulatory property in this plant. The induction of differential T-helper cell immune response appears ideal to overcome immunosuppression as observed in case of lymphatic, filarial Brugia malayi infection which may also be extended to other infections as well.

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.