Loa loa Microfilariae in Skin Snips: Consequences for Onchocerciasis Monitoring and Evaluation in L. loa-Endemic Areas.

The specificity of skin snips for onchocerciasis diagnoses is considered to be almost 100%. Our molecular methods revealed that microfilariae emerging from skin snips collected from highly microfilaremic Loa loa-infected individuals were largely misidentified as Onchocerca volvulus. This has important implications for onchocerciasis diagnostic testing in Loa-endemic areas.

Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi.

N-linked glycosylation is a critical post translational modification of eukaryotic proteins. N-linked glycans are present on surface and secreted filarial proteins that play a role in host parasite interactions. Examples of glycosylated Brugia malayi proteins have been previously identified but there has not been a systematic study of the N-linked glycoproteome of this or any other filarial parasite. In this study, we applied an enhanced N-glyco FASP protocol using an engineered carbohydrate-binding protein, Fbs1, to enrich N-glycosylated peptides for analysis by LC-MS/MS. We then mapped the N-glycosites on proteins from three host stages of the parasite: adult female, adult male and microfilariae. Fbs1 enrichment of N-glycosylated peptides enhanced the identification of N-glycosites. Our data identified 582 N-linked glycoproteins with 1273 N-glycosites. Gene ontology and cell localization prediction of the identified N-glycoproteins indicated that they were mostly membrane and extracellular proteins. Comparing results from adult female worms, adult male worms, and microfilariae, we find variability in N-glycosylation at the protein level as well as at the individual N-glycosite level. These variations are highlighted in cuticle N-glycoproteins and adult worm restricted N-glycoproteins as examples of proteins at the host parasite interface that are well positioned as potential therapeutic targets or biomarkers.

Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi.

Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.

ABC transporters influence sensitivity of Brugia malayi to moxidectin and have potential roles in drug resistance.

Some ABC transporters play a significant role in human health and illness because they confer multidrug resistance (MDR) through their overexpression. Compounds that inhibit the drug efflux mechanism can improve efficacy or reverse resistance. Of the eight described ABC transporter subfamilies, those proteins conferring MDR in humans are in subfamilies A, B, C, and G. In nematodes, transporters in subfamilies B and C are suggested to confer resistance to ivermectin. The Brugia malayi ABC transporter superfamily was examined to assess their potential to influence sensitivity to moxidectin. There was an increase in expression of ABC transporters in subfamilies A, B, C, and G following treatment. Co-administration of moxidectin with inhibitors of ABC transporter function did not enhance sensitivity to moxidectin in males; however, sensitivity was significantly enhanced in females and microfilariae. The work suggests that ABC transporters influence sensitivity to moxidectin and have a potential role in drug resistance.

Infrared light photobiostimulation mediates periodicity in Dirofilaria immitis microfilariae.

Microfilariae (Mfs) of filarial nematode parasites exhibit nocturnal periodicity, with their numbers in peripheral blood peaking at night and decreasing during the day. However, the reason for their appearance at night remains unknown. In this study, in vitro photobiostimulation experiments showed that Mfs exhibited positive phototaxis toward infrared light with lower photon flux densities of infrared light at wavelengths of 890 and 700 nm, in particular, mediating paradoxically higher velocity than intense ones. Microarray analysis revealed that infrared light stimulation influenced gene expression in Mfs and induced significant upregulation of genes, with phosphorylation- and neurogenesis-related genes being highly enriched. Weaker natural infrared beams from the atmosphere only at midnight may induce microfilaria periodicity, and the nature of the periodic pattern is innate and plastic, as demonstrated by artificially changing the light-dark cycle. This is the first report of positive phototaxis toward infrared light in Dirofilaria immitis Mfs. The notable finding is that they moved in union despite the lack of a fluid current inside the container, indicating that infrared light appears to control nocturnal periodicity in D. immitis Mfs. The newly developed culture medium and the adoption of charge-coupled device (CCD) camera and time-lapse VHS videocassette recorder used in this study made possible to be a long observation.

In vitro effect of four herbal plants on the motility of Brugia malayi microfilariae.

BACKGROUND & OBJECTIVE: Disease burden due to lymphatic filariasis is disproportionately high despite mass drug administration with conventional drugs. Usage of herbal drugs in traditional medicine is quite well known but largely empirical. Hence the present study was designed to screen the in vitro antifilarial effect of four herbal plants on Brugia malayi. METHODS: Motility of microfilariae of B. malayi after incubation for 48 h with aqueous/methanol extracts of Vitex negundo L. (roots), Butea monosperma L. (roots and leaves), Ricinus communis L. (leaves), and Aegle marmelos Corr. (leaves) was explored in the concentration range of 20 to 100 ng/ml for possible antifilarial effect by comparing with suitable solvent control. RESULTS: Butea monosperma leaves and roots, Vitex negundo root and Aegle marmelo leaves showed significant inhibition of motility of microfilariae as compared to controls whereas inhibitory activity demonstrated by Ricinus communis L. leaves was not significant. Antifilarial effects imparted by all these extracts were found to be a function of their relative concentrations. Inhibitory concentrations (IC(50)) for the plant extracts with significant antifilarial activity against Brugia malayi microfilariae in in vitro system have been derived to be 82, 83 and 70 ng/ml for Vitex negundo L., Butea monosperma L. and Aegle marmelos Corr. respectively. INTERPRETATION & CONCLUSION: The present study recorded significant antifilarial effect of all plant extracts studied except for Ricinus communis L. leaves and contributes to the development of database for novel drug candidates for human lymphatic filariasis.

C1q enhancement of antibody-dependent granulocyte-mediated killing of nonphagocytosable targets in vitro.

A possible role for C1q in antibody-dependent granulocyte-mediated killing of nonphagocytosable targets was investigated utilizing IgG-dependent granulocyte cytotoxicity directed against microfilariae of Dirofilaria immitis. Granulocyte-mediated killing of microfilariae is enhanced by addition of fresh serum. Lack of C4 did not significantly reduce the observed increase in cytotoxicity. The addition of highly purified monomeric human Clq (0.2 microgram/ml) in the presence of immune IgG resulted in a two- to fivefold enhancement of killing (P less than 0.025). C1q enhancement of killing occurred in the absence of fluid-phase IgG, but killing was significantly less than when both fluid-phase IgG and C1q were present. The effect of C1q was inhibited by the addition of solubilized type I collagen (44-92% inhibition of killing, P less than 0.05). Significant 125I-Clq binding to microfilariae occurred only in the presence of immune IgG. In addition, C1q in concentrations ranging from 0.5 to 2.0 micrograms/ml resulted in a dose-dependent increase in binding of 125I-immune IgG to microfilariae. Finally, when purified C1q was added to preopsonized, washed microfilariae, granulocyte production of superoxide was increased from 0.25 +/- 0.07 to 0.68 +/- 0.07 nm/10(6) cells.10 min (P less than 0.01). These results describe a novel functional role for C1q in enhancement of antibody-dependent cellular cytotoxicity towards nonphagocytosable targets.

Hsp90 is essential in the filarial nematode Brugia pahangi.

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.

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.

Isolation and characterization of endochitinase and exochitinase of Setaria cervi.

Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72±0.25 nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40±0.25 nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65-70 kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3±0.18 µM.

Poorer NF-kappa B signaling by microfilariae in macrophages from BALB/c mice affects their ability to produce cytotoxic levels of nitric oxide to kill microfilariae.

Upon activation with microfilariae (mf), macrophages from C57Bl/6 mice showed higher nuclear factor-kappa B (NF-kappa B) but lower activating protein 1 DNA-binding activity as compared to BALB/c macrophages. The C57Bl/6 macrophages produced cytotoxic levels of nitric oxide (NO) to kill Setaria cervi mf as compared to BALB/c macrophages. Inhibition of the NF-kappa B signal by pyrrolidine dithiocarbamate (PDTC) blocked NO production and microfilaricidal activity of C57Bl/6 macrophages and inclusion of the exogenous NO generator (SNP) in the PDTC treated C57Bl/6 macrophage cultures induced mf cytotoxicity. These results underscore that the NF-kappa B signal (induced in response to mf) is important for the NO-mediated microfilaricidal activity of macrophages.

The expression of small heat shock proteins in the microfilaria of Brugia pahangi and their possible role in development.

Development of the microfilariae of Brugia pahangi in the mammalian host is blocked until uptake by a mosquito vector when the developmental cycle is re-initiated. Comparison of the profile of polypeptides labelled in microfilariae cultured at mammalian temperature (37 degrees C) or mosquito temperature (28 degrees C) revealed a complex of low-molecular-weight proteins (18 kDa and 22-24 kDa) synthesized only in microfilariae at 37 degrees C. The synthesis of these proteins was also induced by transfer of microfilariae to 41 degrees C (i.e., heat shock conditions), suggesting that these are heat shock proteins. The expression of the small heat shock proteins in the Brugia life cycle is developmentally regulated, as they are not observed in the mature adult female. Their synthesis is strictly temperature dependent and is repressed upon transfer of the microfilariae to 28 degrees C.

Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development.

Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.

Transglutaminase-catalyzed incorporation of host proteins in Brugia malayi microfilariae.

Recently, we have characterized and purified a novel transglutaminase (pTGase) from adults of the filarial worms Brugia malayi. pTGase-catalyzed reactions seem to play an essential role during in utero growth and development of microfilariae. The results presented here demonstrate that exudates from the peritoneal cavity of jirds, the site where adult worms of B. malayi reside and produce microfilariae, contain several host proteins that can serve as substrates in pTGase-catalyzed reactions. The peritoneal exudate proteins are avidly taken up by adult female worms in vitro and incorporated into the developing microfilariae. Among the several host proteins that were crosslinked, a 68-kDa molecular weight protein (p68) was found to be the major protein taken up by the parasites. Following uptake by the parasites, the peritoneal exudate proteins are crosslinked to form high molecular weight aggregates, that are subsequently incorporated into in utero developing embryos and microfilariae. The cross-linking of host proteins was, however, inhibited by monodansylcadaverine (MDC), a competitive inhibitor of pTGase. Antibodies raised against the jird peritoneal exudate proteins strongly immunoreacted with a 68-kDa protein in adult worms and microfilariae extracts but not with infective-stage larvae (L3) of B. malayi. These results suggest that pTGase is involved in covalent incorporation of host proteins (such as p68) into developing embryos and microfilariae of B. malayi.

Diethylcarbamazine inhibits endothelial and microfilarial prostanoid metabolism in vitro.

Diethylcarbamazine (DEC) rapidly lowers the number of microfilariae in the peripheral circulation. The mechanism of action is unknown, but may involve alterations of arachidonic acid metabolism in vascular tissues. We studied the effects of DEC on arachidonic acid metabolism by bovine pulmonary arterial endothelium monolayers, human platelets and Brugia malayi microfilariae. DEC at a concentration of 2.5 microM, a level achieved in vivo, rapidly decreased prostacyclin, prostaglandin E2 and thromboxane B2 release from endothelial monolayers by 78% (P less than 0.001), 57% (P = 0.05), and 75% (P less than 0.05), respectively. High-pressure liquid chromatography of extracts of endothelial monolayers incubated with DEC showed similar inhibition of these cyclooxygenase pathway products, but exposure to the drug did not result in formation of new eicosanoids. DEC did not inhibit endothelial phospholipase A2-dependent release of arachidonate from membrane stores, whereas prostaglandin H2 synthase activity (cyclooxygenae, EC 1.14.99.1) was reduced to a degree similar to that effected by acetylsalicylic acid. Microfilarial but not platelet synthesis of cyclooxygenase products was also reduced by DEC. These data suggest that the mechanism by which DEC lowers the level of microfilariae in the circulation may in part involve its effects on host endothelial and parasite eicosanoid production.

Lectin binding to larval stages of Brugia patei.

Surface properties of microfilariae (mf) and infective larvae of Brugia patei were investigated to compare them to previous studies with the other brugian species. Of all the lectins tested, only wheat germ agglutinin (WGA) binds to the sheath surface of mf indicating the presence of N-acetyl-D-glucosamine as a major surface carbohydrate. However, cuticle of infective larvae failed to show binding of these lectins. Enzyme treatment of mf with N-acetyl-D-glucosaminidase and L-fucosidase has exposed D-mannose, N-acetyl-D-galactosamine and L-fucose on the sheath surface. The binding of lectins to intact mf and to enzyme-treated mf appeared to be specific as pretreatment with specific inhibitory sugars completely abolished the binding activity. This is the first study conducted with this filarial parasite and it established that B. patei is similar to other species of Brugia but differs from Wuchereria in its surface-lectin binding properties.

Uptake of amino acids and glucose by microfilariae of Dirofilaria immitis in vitro.

The uptake of amino acids and glucose by microfilariae of Dirofilaria immitis maintained for 8 days in vitro was studied. The quantity of glutamic acid, histidine, lysine, alanine, tryptophan, and cystine in the medium did not change. Isoleucine, serine, valine, phenylalanine, tyrosine, methionine, and cystathionine decreased slightly; both asparatic acid and leucine decreased considerably. Glutamine, threonine, and proline decreased markedly, while glycine increased to some extent. Ornithine increased and arginine decreased; however, their changes were smaller than the changes of respective amino acids in the control medium free of microfilariae. Glucose decreased markedly.

Release of prostaglandin E2 by microfilariae of Wuchereria bancrofti and Brugia malayi.

To elucidate the local release of immunomodulatory prostaglandins by intravascular filarial parasites, the formation of prostaglandin E2 (PGE2) was examined in individual microfilariae of Wuchereria bancrofti and Brugia malayi. Following incubation of living microfilariae immobilized in an agar matrix, prostaglandins released by the parasites were fixed by carbodiimide and localized by indirect immunofluorescence. Prostaglandin E2 was specifically detected around the entire surface of microfilariae with anti-PGE2 antiserum, but not with control nonimmune or PGE2 affinity-immunoadsorbed antiserum. These results provide direct evidence that individual microfilariae of W. bancrofti as well as B. malayi release prostaglandins into their microenvironment. The release of PGE2 by these intravascular parasites may modulate host leukocyte responses, and thereby contribute to the immune defects observed in infected humans with peripheral microfilaremia.

Lectin-binding characteristics of Wuchereria bancrofti microfilariae.

The binding of 10 different lectins to the surface of microfilariae of Wuchereria bancrofti has been investigated. Wheat germ agglutinin (WGA) and Helix pomatia lectin (HPA) bound specifically to the sheathed microfilariae indicating the presence of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine respectively on the surface. Exsheathed microfilariae did not react with any of the lectins. Treatment of sheathed microfilariae with proteases resulted in increased binding of WGA and HPA. Such treated microfilariae showed a weak binding of Concanavalin A (Con A), and lectins of lentil (LCH) and of Limulus polyphemus (LPA). Sheathed microfilariae incubated with sera of people living in endemic zones of filariasis but with no apparent evidence of infection (endemic normals), or with sera of chronic elephantiasis patients, or with their respective gamma globulin fractions, bound Con A and LCH. These lectins bound weakly to exsheathed microfilariae under the same conditions. Binding was due to the mannose components of the specific immunoglobulins of the sera which coated the microfilariae. However, microfilariae when incubated with sera or their globulin fractions from non-endemic normals (NEN), or from microfilarial carriers, did not bind Con A and LCH, suggesting that specific immunoglobulins were neither present in NEN sera nor in significant amounts in sera of microfilarial carriers.

An in vitro radiolabel uptake viability assay for Onchocerca microfilariae.

A radiolabel uptake viability assay for Onchocerca cervicalis using [3H]2-deoxy-D-glucose in Hanks' balanced salt solution, pH 7.5, at 30 C is described and compared to the traditional visual motility assay. A correlation of r = 0.92 between the assays was found, with the radiolabel uptake method apparently a more sensitive indicator of microfilarial viability.