S.K. Joseph

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi.

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC(50)) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT>80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin>flavone=hesperetin>rutin>naringin>chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.

Immunization with inflammatory proteome of Brugia malayi adult worm induces a Th1/Th2-immune response and confers protection against the filarial infection

Mastomys coucha and jirds (Meriones unguiculatus) were immunized with four cytokine-stimulating SDS-PAGE resolved fractions F5 (68-84 kDa), F6 (54-68 kDa), F10 (38-42 kDa) and F14 (20-28 kDa) of Brugia malayi adult worm to determine which of these fractions has the potential to influence the establishment of subsequently introduced B. malayi infection in the animals. The proteins in the fractions were analyzed by 2DE and MALDI-TOF. Immunization with F6 suppressed the establishment of third stage larva (L(3)) initiated infection in M. coucha (64%; P<0.01) and jird (42%; P<0.01). Survival of intraperitoneally implanted adult worms in M. coucha was lowered by F6 (72%; P<0.01) and F14 (66%; P<0.05) but not by F5 and F10. Immunization with F6 intensely upregulated both Th1 (IFN-gamma, TNF-alpha, IL-1 beta, IL-2, IL-6, IgG1, IgG2a and lymphoproliferation) and Th2 (IgG2b and IL-10) responses and NO release. Immunostimulatory proteins HSP60, intermediate filament protein, and translation elongation factor EF-2 were identified in F6 fraction by 2DE and MALDI. The findings suggest that F6 protects the host from the parasite via Th1/Th2 type responses and thus holds promise for development as a vaccine.

Plant Products in the Treatment and Control of Filariasis and Other Helminth Infections and Assay Systems for Antifilarial/Anthelmintic Activity

Lymphatic filariasis, onchocerciasis, loaisis, and other helminth infections cause serious health problems especially in resource-limited tropical and subtropical developing countries of the world, and more than 2 billion people are infected with at least one helminth species. From times immemorial, man looked up to the plant kingdom in search of anthelmintics, antifilarials, and remedies for parasite-induced health problems. Although more than 50 % of drugs in modern medicine are derived from plants or leads from plants, a success story of plant-based anthelminthics or antifilarials is yet to be told. In the last 5 decades, more than 100 plant products were reported to be beneficial in the treatment or control of these parasitic infections but they could not be developed into viable drugs for a variety of reasons. This review focuses on the plant products reported to be useful in the control and treatment of human helminth infections with the main emphasis on filariasis and the in vitro and in vivo systems available for assaying anthelmintic activity.

Sensitization with anti-inflammatory BmAFI of Brugia malayi allows L3 development in the hostile peritoneal cavity of Mastomys coucha

Filarial parasites survive by inducing tolerance in host but the antigens and mechanisms involved are not clear. Recently we found that BmAFI, a Sephadex G-200 eluted fraction of Brugia malayi adult worm extract, stimulates IL-10 release from THP-1 cells. In the present study, we determined the SDS-PAGE profile of BmAFI and infective 3rd stage larva (L3), investigated the effect of pre-sensitization of host with BmAFI on the survival and development of L3 in the non-permissive peritoneal cavity (p.c.) of the permissive host Mastomys coucha and in the p.c. of non-permissive Swiss mice, and studied immunological correlates for the observed effects. The parasite development and burden in p.c., was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-β, IL-4, IL-10, IL-13, IFN-γ and NO, cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard subcutaneous (s.c.) route to assess whether sensitization enhances the intensity of infection. BmAFI-sensitization permitted survival of L3 and their development to adult stage by day 60 p.i. in the p.c. of M. coucha; in non-sensitized animals L3 could molt to L4 only and no parasite could be recovered beyond day 30 p.i. In M. coucha that received infection by s.c. route, pre-sensitization with BmAFI enhanced the microfilaraemia and adult worm recovery. In sensitized Swiss mice L3 could successfully molt to L4 in p.c. with improved recovery of parasite. BmAFI sensitization upregulated TGF-β and IL-10 release, IgG1 and IgG2b levels, GATA-3 and CTLA-4 mRNA expression, suppressed the cellular proliferative response and downregulated Con A stimulated response, IgE, IL-13, IFN-γ and NO responses. Immunoblot analysis showed that the BmAFI antiserum also strongly reacts with some L3 molecules. The results show, for the first time, that sensitization with the anti-inflammatory BmAFI which shares some of its molecules with those in L3, facilitates parasite survival in the non-permissive p.c. of the permissive host M. coucha, render a non-permissive Swiss mouse partially permissive to infection and enhances parasite load in M. coucha receiving the infection through permissive s.c. route by evoking a modified Th2 type of response and anti-inflammatory milieu. In conclusion, the findings suggest that the anti-inflammatory BmAFI fraction facilitates survival of B. malayi infection even in non-permissive environment.

Influence of Brugia malayi life stages and BmAFII fraction on experimental Leishmania donovani infection in hamsters.

The influence of live Brugia malayi parasites and a Sephadex G-200 fraction of the adult parasite extract (BmAFII) on the progression of Leishmania donovani infection was studied. Inbred hamsters were first infected with B. malayi infective 3rd stage larvae (L3), adult worms or microfilariae (mf), and then with L. donovani amastigotes (Ld), or vice versa or received both the infections simultaneously; a group of animals were first immunized with BmAFII and then infected with Ld. L. donovani parasite burden was determined between 17 and 19 days post amastigote challenge (p.a.c.) and, in case of immunized animals, between 32 and 35 days p.a.c also. Nitric oxide (NO) release from peritoneal macrophages and cellular proliferative responses of lymphnode cells were assessed in BmAFII-immunized animals given leishmania infection or no infection. Leishmanial parasite burden was significantly reduced in animals exposed to filarial L3 before amastigote inoculation and in animals given filarial adult worms after or together with amastigotes. Prior immunization of leishmania-infected animals with BmAFII also reduced the leishmanial parasite burden (17-19 days p.a.c.: >90%; 32-35 days p.a.c.: 60%). These animals showed upregulation of NO release and cellular proliferative responses to promastigote antigen or BmAFII stimulation in vitro. The findings show, for the first time, that B. malayi L3/adult worms or immunization with BmAFII inhibits progression of L. donovani infection in hamsters and this is associated with upregulation of NO and lymphocyte proliferative responses indicating that Th1 response might be responsible for this.

Humoral and cell-mediated immune-responses after administration of a single-shot recombinant hepatitis B surface antigen vaccine formulated with cationic poly(l-lactide) microspheres

The present investigations were aimed to compare the humoral and cell-mediated immune responses between recombinant hepatitis B surface antigens (HBsAg) adsorbed L-PLA microspheres (Ms) vaccine (single-shot) and marketed alum-HBsAg vaccine (two-doses). The blank cationic (cetyltrimethyammoniumbromide) microspheres were prepared by the double emulsion (w/o/w) solvent evaporation technique. The HBsAg was adsorbed onto the surface of blank cationic microspheres. These microspheres were characterized in vitro for their size, shape, adsorption-efficiency, in-process stability, and HBsAg release studies. Specific humoral immune responses (IgM and IgG) and cell-mediated immune responses (cellular-proliferation) assay including release of interferon-gamma (IFN-γ), interleukin-2 (IL-2), and nitric oxide (NO) from host’s cells stimulated with HBsAg or lipopolysaccharide (LPS)/ concanavalin A (con A) in-vitro were determined. Based on these findings, it was concluded that the single injection (using subcutaneous-route) of the polymeric microspheres produced better immune response (both humoral and cell-mediated) than two injections of a conventional alum-HBsAg vaccine. These data demonstrate high potential of polymeric microspheres for their use as a carrier adjuvant for hepatitis B vaccine.

Cross reactive molecules of human lymphatic filaria Brugia malayi inhibit Leishmania donovani infection in hamsters

Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.

Humoral and cell-mediated immune responses elicited by poly (dl-lactide) adjuvanted filarial antigen molecules

Abstract Context: In our recent studies, Brugia malayi molecules have shown interesting immune-stimulating and immune-suppressive properties. Among these, F6 a pro-inflammatory (54–68 kDa) SDS-PAGE resolved fraction of the parasite when administered with Freund’s complete/incomplete adjuvant in animals, elicited both Th1 and Th2 type immune responses and protects the host from filarial parasite. Objective: The present study was aimed at developing biodegradable microspheres for filarial antigenic protein molecules and to investigate the immunoadjuvanticity of microspheres (Ms)-loaded F6 molecules. Materials and methods: Poly-lactide microspheres (DL-PLA-Ms) were prepared using double emulsification and solvent evaporation method; and studied their size, shape, antigen adsorption efficiency, in-process stability, and antigen release profiles. F6 and B. malayi adult worm (BmA: ∼17 to 180 kDa) protein molecules adsorbed on the Ms were administered in a single shot into Swiss mice, subcutaneously, and investigated their immunoadjuvant effect and compared with one/two doses-schedule of plain F6/BmA. Results: Immunization with F6/BmA-loaded DL-PLA-Ms resulted in upregulation of cellular proliferation, IFN- γ, TNF-α and NO release from host’s cells stimulated with F6/BmA or LPS/Con A, IgG, IgG1 and IgG2a levels. These responses were well comparable with the responses produced by two doses of plain BmA/F6. Discussion and conclusion: In conclusion, a single dose of DL-PLA-Ms-F6 induced predominantly Th1 immune responses and well comparable with two doses of plain F6. This is the first ever report on potential of DL-PLA-Ms as adjuvant for filarial immunogen.

Anti-inflammatory BmAFI of Brugia malayi modulates IgE, histamine and histamine receptor responses in Mastomys coucha

We recently reported that BmAFI, an anti-inflammatory fraction of Brugia malayi adult worm supports parasite development in the hostile peritoneal cavity (p.c.) of Mastomys coucha through a modified Th2 type of response that includes IL-13 and IgE response and anti-inflammatory IL-10 cytokine milieu. In the present study we investigated IgE related responses such as histamine release and modulation of histamine receptors 1 and 2 (HR1 and HR2) by presensitization with BmAFI of M. coucha infected with B. malayi. Sensitization with BmAFI alone enhanced IgE, histamine and HR2, but decreased HR1. Exposure of these animals to infection produced an IgE response that was inversely related to the parasite burden, and decreased histamine conc., and HR1 and HR2 expression. However, there was an early small increase in HR1 expression for a short period after exposure to infection. As expected, BmAFI sensitization supported parasite survival and development in the hostile p.c. of the host. These findings further establish that BmAFI decreases inflammatory/Th1 response and modulates Th2 responses to favour survival and development of the parasite in the hostile p.c. of the host and that IgE and histamine play an important role in this.