Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Antibody and cellular immune responses to microfilarial antigens in ferrets experimentally infected with Brugia malayi

Eleven of 15 ferrets experimentally infected with Brugia malayi became amicrofilaremic after a brief patency; only four ferrets remained patent after 6 months of infection and two of these ferrets developed a high, persistent microfilaremia. Blastogenic responses of peripheral blood lymphocytes to antigens of microfilariae (mf), assayed in vitro, demonstrated an antigen sensitivity at prepatent, patent and postpatent periods of infection. Lymphocytes from ferrets with high microfilaremia had elevated background responses in culture which were directly correlated with the number of circulating mf. This background response was attributed to antigenic stimulation by mf present in the lymphocyte cultures; addition of mf to cultures of lymphocytes from postpatent ferrets induced responses equivalent to those observed in microfilaremic ferrets. Lymphocyte responses to the mitogen, concanavalin A, did not differ significantly among microfilaremic, amicrofilaremic and uninfected ferrets. Antibody in IgG to antigens of mf measured by ELISA and by immunoblots from SDS-PAGE showed similar patterns of response in ferrets which became amicrofilaremic and in the few ferrets which remained microfilaremic. prausnitz-Kustner tests demonstrated no consistent differences in titers to microfilarial antigens between patent and amicrofilaremic ferrets. The results suggest a high level of immune responsiveness to antigens of mf in infected ferrets with no evidence of immunosuppression associated with prolonged microfilaremia or of major changes in immune responses with development of amicrofilaremic infections.     

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.     

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.     

Brugia malayi: arachidonic acid uptake into lipid bodies of adult parasites

Lipid bodies are non-membrane bound intracellular organelles, which have been recognized morphologically in a diversity of mammalian and nonmammalian cells, but are of uncertain function. In mammalian cells, in addition to serving as a storage site of cholesterol and triglyceride, lipid bodies can be a repository of esterified arachidonic acid. Adult worms of the human filarial parasite Brugia malayi have been found to esterify exogenous [3H]arachidonic acid into parasite phospholipids and neutral lipids. Electron microscopic autoradiography demonstrated that [3H]arachidonate was preferentially incorporated into filarial lipid bodies. The dominant incorporation of arachidonate into lipid bodies of a nematode establishes that lipid bodies are a site of arachidonic acid accumulation in nonmammalian, as well as mammalian, cells.     

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.     

High dietary fish oil alters the brain polyunsaturated fatty acid composition

Feeding adult rats a 17% corn-oil diet for 8 weeks did not change brain polyunsaturated fatty acids (PUFA) compared to rats fed 2.2% corn oil (with 2.2% lard added). When the corn-oil diet was supplemented with 14.5% cod liver oil or 12.5% salmon oil, the fatty acid composition of brain PUFA was significantly altered, even if alpha-tocopherol was added to the salmon-oil diet. Comparing salmon-oil- and cod-liver-oil-fed animals with corn-oil-fed animals, arachidonic acid 22:4(n-6) and 22:5(n-6) were reduced, and 20:5(n-3), 22:5(n-3) and 22:6(n-3) were increased. Liver fatty acids were also significantly altered. Thus, the brain is not protected against a large excess of very-long-chain n-3 PUFA, which increase n-3/n-6 ratio and could lead to abnormal function, and which might be difficult to reverse.     

Evolutionary modifications of human milk composition: evidence from long-chain polyunsaturated fatty acid composition of anthropoid milks

Brain growth in mammals is associated with increased accretion of long-chain polyunsaturated fatty acids (LCPUFA) in brain phospholipids. The period of maximum accumulation is during the brain growth spurt. Humans have a perinatal brain growth spurt, selectively accumulating docosahexaenoic acid (DHA) and other LCPUFA from the third trimester through the second year of life. The emphasis on rapid postnatal brain growth and LCPUFA transfer during lactation has led to the suggestion that human milk LCPUFA composition may be unique. Our study tests this hypothesis by determining fatty acid composition for 11 species of captive anthropoids (n = 53; Callithrix jacchus, Cebus apella, Gorilla gorilla, Hylobates lar, Leontopithecus rosalia, Macaca mulatta, Pan troglodytes, Pan paniscus, Pongo pygmaeus, Saimiri boliviensis, and Symphalangus syndactylus). Results are compared to previously published data on five species of wild anthropoids (n = 28; Alouatta paliatta, Callithrix jacchus, Gorilla beringei, Leontopithecus rosalia, and Macaca sinica) and human milk fatty acid profiles. Milk LCPUFA profiles of captive anthropoids (consuming diets with a preformed source of DHA) are similar to milk from women on a Western diet, and those of wild anthropoids are similar to milk from vegan women. Collectively, the range of DHA percent composition values from nonhuman anthropoid milks (0.03–1.1) is nearly identical to that from a cross-cultural analysis of human milk (0.06–1.4). Humans do not appear to be unique in their ability to secrete LCPUFA in milk but may be unique in their access to dietary LCPUFA.     

Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis

Background

Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA.

Methodology/Principal Findings

The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C₂₀ and C₂₂ polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3.

Conclusions

The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.     

The influence of dietary docosahexaenoic acid and arachidonic acid on central nervous system polyunsaturated fatty acid composition

Numerous studies on perinatal long-chain polyunsaturated fatty acid nutrition have clarified the influence of dietary docosahexaenoic acid (DHA) and arachidonic acid (ARA) on central nervous system PUFA concentrations. In humans, omnivorous primates, and piglets, DHA and ARA plasma and red blood cells concentrations rise with dietary preformed DHA and ARA. Brain and retina DHA are responsive to diet while ARA is not. DHA is at highest concentration in cells and tissues associated with high energy consumption, consistent with high DHA levels in mitochondria and synaptosomes. DHA is a substrate for docosanoids, signaling compounds of intense current interest. The high concentration in tissues with high rates of oxidative metabolism may be explained by a critical role related to oxidative metabolism.     

Brugia malayi: transient transfection by microinjection and particle bombardment

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

Brugia malayi: establishment in inbred and outbred strains of mice

Immunocompetent mouse model for human filarial parasite Brugia malayi is urgently required in view of the paucity of commercial reagents for other susceptible rodent viz. mastomys and gerbil. Genes within the major histocompatibility complex have been reported to influence the susceptibility of mouse to helminth parasites. Attempts have therefore been made in the present investigation to experimentally infect various inbred strains of mice viz. NZB/BINJ, BALB/c, AKR, C(3)H, and SJL/J with H-2 haplotype (H-2: d, d, k, k, s, respectively) and outbred strains of mice viz. Parks and Swiss. Findings indicate that susceptibility of mice to B. malayi is strain associated. This is the first report on the successful completion of full developmental cycle of subperiodic B. malayi in NZB/BINJ, an immunocompetent mouse strain. In some of the other strains, partial development or low degree of establishment of worms was observed.     

Brugia malayi: patterns of expression of surface-associated antigens.

Patterns of expression of surface-associated antigens were analyzed in the filarial nematode Brugia malayi immediately prior, and during development in the vertebrate host. Two surface-associated protein molecules, i.e., accessible to surface radioiodination and soluble in aqueous buffers, were investigated: Mrs 29-30,000 and 16,000, both of which are antigenic in infected animals. The Mr 29-30,000 glycoprotein is expressed in a surface-associated manner by adult worms and by fourth-stage larvae, but is not detectable in preparasitic third-stage larvae. The 16,000 component, which appears not to be glycosylated, is surface-associated in adult worms and fourth-stage larvae. In contrast to the 29-30,000 glycoprotein, the 16,000 protein is also expressed both by pre- and postparastic third-stage larvae. However, it becomes surface-associated only after infection. Thus, immediately prior, and during development within the vertebrate host, B. malayi displays at least two different patterns of expression of surface-associated antigens: (i) de novo, intiated either immediately after infection (phase specific) or during genesis of the fourth-stage larva (stage specific); (ii) continuous, but with phase-dependent surface exposure of previously cryptic antigens, during the transition from intermediate to definitive host.     

Analysis of polyunsaturated fatty acid composition of Strongyloides ratti in relation to development

The effect of linolenic acid (C18:3 omega 3) on the development of Strongyloides ratti first-stage larvae (L1) in culture was studied. The fatty acid composition of S. ratti free-living generations was analyzed by gas chromatography. L1 had abundant linoleic acid (C18:2 omega 6) but its proportion decreased with development. On the contrary, eicosapentaenoic acid (C20:5 omega 3) and C20:4 omega 3 were prominent in the filariform larva (L3). Because C20:5 omega 3 is generally synthesized from C18:3 omega 3 via C20:4 omega 3, the high ratio of C20:5 omega 3/C18:3 omega 3 of L3 in all the free-living generations suggested that polyunsaturated fatty acid metabolism, particularly the omega-3 series, and eicosanoids produced had important roles in the development of S. ratti L1.