Molting Of L3 Research Articles

Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis.

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Onchocerca volvulus Molting Inhibitors Identified through Scaffold Hopping.

The anthelmintic closantel has shown promise in abrogating the L3 molting of Onchocerca volvulus, the causative agent of the infectious disease onchocerciasis. In our search for alternative scaffolds, we utilized a fragment replacement/modification approach to generate novel chemotypes with improved chitinase inhibitory properties. Further evaluation of the compounds unveiled the potential of urea-tropolones as potent inhibitors of O. volvulus L3 molting.

Dual protonophore-chitinase inhibitors dramatically affect O. volvulus molting.

The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode’s developmental process.

Design, Synthesis, and Biological Activities of Closantel Analogues: Structural Promiscuity and Its Impact on Onchocerca volvulus

Onchocerciasis, or river blindness, is a neglected tropical disease that affects more than 37 million people worldwide, primarily in Africa and Central and South America. We have disclosed evidence that the larval-stage-specific chitinase, OvCHT1, may be a potential biological target for affecting nematode development. On the basis of screening efforts, closantel, a known anthelmintic drug, was discovered as a potent and highly specific OvCHT1 inhibitor. Originally, closantel's anthelmintic mode of action was believed to rely solely on its role as a proton ionophore; thus, the impact of each of its biological activities on O. volvulus L3 molting was investigated. Structure-activity relationship studies on an active closantel fragment are detailed, and remarkably, by use of a simple salicylanilide scaffold, compounds acting only as protonophores or chitinase inhibitors were identified. From these data, unexpected synergistic protonophore and chitinase inhibition activities have also been found to be critical for molting in O. volvulus L3 larvae.

The Onchocerca volvulus Cysteine Proteinase Inhibitor, Ov-CPI-2, Is a Target of Protective Antibody Response That Increases with Age

BackgroundDespite considerable efforts, a suitable vaccine against Onchocerca volvulus infection has remained elusive. Herein, we report on the use of molecular tools to identify and characterize O. volvulus antigens that are possibly associated with the development of concomitant immunity in onchocerciasis.Methodology/Principal FindingsThird-stage larvae (L3) and molting L3 (mL3) O. volvulus stage-specific cDNA libraries were screened with a pool of sera from chronically infected patients who had likely developed such immunity. The 87 immunoreactive clones isolated were grouped into 20 distinct proteins of which 12 had already been cloned and/or characterized before and 4 had been proven to be protective in a small O. volvulus animal model. One of these, onchocystatin (Ov-CPI-2), a previously characterized O. volvulus cysteine proteinase inhibitor was, overall, the most abundant clone recognized by the immune sera in both the L3 and mL3 cDNA libraries. To further characterize its association with protective immunity, we measured the IgG subclass and IgE class specific responses to the antigen in putatively immune (PI) and infected (INF) individuals living in a hyperendemic area in Cameroon. It appeared that both groups had similar IgG3 and IgE responses to the antigen, but the INF had significantly higher IgG1 and IgG4 responses than the PI individuals (p<0.05). In the INF group, the IgG3 levels increased significantly with the age of the infected individuals (r = 0.241; p<0.01). The IgG1 responses in the INF were high regardless of age. Notably, culturing L3 in vitro in the presence of anti-Ov-CPI-2 monospecific human antibodies and naïve neutrophils resulted in almost complete inhibition of molting of L3 to L4 and to cytotoxicity to the larvae.Conclusions/SignificanceThese results add to the knowledge of protective immunity in onchocerciasis and support the possible involvement of anti-Ov-CPI-2 IgG1 and/or IgG3 cytophilic antibodies in the development of protective immunity in the PI and the INF. The results further support the consideration of Ov-CPI-2 as a leading target for an anti-L3 vaccine.

Brugia malayi: Effects of radiation and culture on gene expression in infective larvae

Third-stage infective larvae (L3i) of Brugia malayi are developmentally arrested in mosquitoes but must quickly adapt to a new environment when they enter mammalian hosts to initiate infections. These changes can be studied by in vitro culture of L3 (L3c) under conditions that permit molting of L3–L4. Irradiated L3 (L3ir) have stunted growth and limited lifespan in mammalian hosts, and they induce high levels of immunity to challenge infections in animal models. This study explored differences in gene expression in L3i, L3c and L3ir by expressed sequence tag EST generation and qRT-PCR. 2506 ESTs generated from cDNA libraries constructed from L3i, L3c and L3ir were grouped into 1309 gene clusters. Despite extensive prior sampling from B. malayi (>22,000 ESTs in dbEST), 73% of the L3 clusters described here are novel. Sixty-three percentage of the clusters have homology to proteins from other species including 187 specific to nematodes and 141 that have to date only been described in non-nematode species. The transcript levels of 62 candidates for up- or down-regulation in L3i, L3c and L3ir based on EST frequencies were evaluated by qRT-PCR. Twenty-eight were confirmed to have ≥3-fold differences in expression. Genes coding for proteins believed to be involved in establishment of infection, host adaptation and targets of protective immunity were confirmed to have higher expression in L3i than in L3c. Some of the genes that were down-regulated in L3c were highly expressed in L3ir. This study provides an improved description of the adaptations that accompany the transition from L3i to L3c and the special ability of L3ir to induce protective immunity.

Effects of gamma radiation on Brugia malayi infective larvae and their intracellular Wolbachia bacteria

Prior studies have shown that irradiated filarial larvae are developmentally stunted but capable of inducing partial immunity to filariasis in animals. The mechanisms for these effects are poorly understood. Recent studies suggest that intracellular Wolbachia bacteria are necessary for the normal development, reproduction and survival of filarial nematodes. The purpose of this study was to examine the effects of irradiation on Wolbachia in Brugia malayi infective larvae (L3) and on L3 development. The L3 were exposed to 0, 25, 35, 45, 55, 65 or 75 krad of gamma irradiation from a (137) Cesium source and cultured in vitro at 37 degrees C in NCTC/IMDM medium with 10% FCS for 12 days. Irradiation prevented molting of L3 to the L4 stage in a dose-dependent manner. Electron microscopy studies showed that irradiation damaged Wolbachia (25 krad) or cleared them from worm tissues (45 krad). In addition, majority of the irradiated L3s failed to develop the L4 cuticle. Real-time PCR studies showed that irradiation reduced Wolbachia DNA in worm tissues. Parallel in vivo studies confirmed decreased development of irradiated L3 in jirds, with associated effects on Wolbachia. Jirds injected s.c with normal L3 developed antibodies to Wolbachia surface protein (wsp) shortly after the onset of microfilarial patency. In contrast, jirds injected with irradiated L3 did not develop microfilaremia or antibodies to wsp. Additional studies are needed to test the hypothesis that irradiation retards growth and development of filarial L3 by killing Wolbachia.

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.

Inhibitors of the lipoxygenase pathway block development of Brugia malayi L3 in vitro.

Brugia malayi L3 molt to the L4 stage in serum-free cultures supplemented with arachidonic, linoleic, or linolenic acids and the basidiomycetous yeast Rhodotorula minuta. These fatty acids are capable of entering the eicosanoid pathway of arachidonate metabolism, the pathway responsible for generating a number of biologically active mediators, including prostaglandins, leukotrienes, and lipoxins. To determine whether this pathway was required for L3 development, we added dual inhibitors of cyclooxygenase and lipoxygenase to in vitro cultures containing B. malayi L3. These compounds significantly inhibited L3 molting. To evaluate whether 1 or both of these pathways of arachidonate metabolism were involved in molting, we tested drugs inhibiting either cyclooxygenase or lipoxygenase. Lipoxygenase inhibitors blocked L3 molting, whereas cyclooxygenase inhibitors did not. To assess whether enzymes operating downstream of lipoxygenase were also involved in L3 molting, we added inhibitors of enzymes involved in leukotriene synthesis and found they were also capable of preventing development. We tested the same inhibitor panel on Dirofilaria immitis L3. A single lipoxygenase inhibitor and inhibitors of 2 different enzymes operating downstream of lipoxygenase disrupted D. immitis development. These results demonstrate that a lipoxygenase pathway product is required for molting of the infective stage larvae of filarial parasites.

Cloning of a Cysteine Protease Required for the Molting of Onchocerca volvulus Third Stage Larvae

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 microM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae.

Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.

Evaluation of Different Medium Supplements for In vitro Cultivation of Brugia malayi Third-Stage Larvae

Growth and development of Brugia malayi (Nematoda: Filarioidea) third-stage larvae (L3) were compared in 5 medium supplements. The basic culture medium (NI) consisted of a 1:1 (v/v) mixture of NCTC-135 and Iscove's modified Dulbecco's medium, an antibiotic/antimycotic mixture, and 1 of the following 5 supplements: 25 mg/ml bovine albumin fraction-V (BAF), 10% fetal bovine serum (FBS), 10% commercially obtained human serum (CHS), 10-15% pooled human serum from hospital patients (PHS), and 10-15% human serum from a single individual (SHS). Cultures were maintained at 37 C in an atmosphere of 5% CO2 in air. NI-BAF and NI-CHS did not support molting of L3 to fourth-stage larvae (L4), whereas NI-FBS, NI-PHS, and NI-SHS did support molting of L3 to L4 but only the larvae in NI-SHS attempted the fourth molt. Growth and development of in vitro larvae in NI-PHS and NI-SHS were comparable to that observed in jirds for the first 28 days, after which the in vitro larvae lagged behind in vivo larvae. Optimal growth and development may be dependent on certain as yet unidentified components of specific human serum.