ABSTRACT
The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.
Subject(s)
Dipetalonema/immunology , Dipetalonema/physiology , Helminth Proteins/genetics , Helminth Proteins/immunology , Recombinant Proteins/immunology , Amino Acid Sequence , Animals , Circular Dichroism/methods , Cross Reactions , Dipetalonema/genetics , Electrophoresis, Polyacrylamide Gel , Female , Glycosylation , Helminth Proteins/chemistry , Helminth Proteins/metabolism , Immunoglobulin G/metabolism , Mice , Mice, Inbred BALB C , Mutagenesis, Site-Directed/methods , Phosphorylcholine/chemistry , Phosphorylcholine/metabolism , Pichia/genetics , Polymerase Chain Reaction/methods , Protein Structure, Secondary/physiology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Time Factors , Ultracentrifugation/methodsABSTRACT
Octopamine acts as an important neurotransmitter and neuromodulator in arthropods, mollusks, and nematodes. In mammals, however, no definite function for this amine has yet been described. By virtue of this difference in the neurophysiological requirement of the mammalian host and nematodes, octopamine offers good opportunity for exploring this area deeply with a view to identify a unique target for filarial chemotherapy. Results of the present study indicated that Acanthocheilonema viteae, the rodent filarial parasite, utilized tyrosine as a precursor for producing octopamine and some other biogenic amines. Octopamine exhibited specific saturable binding with the membrane prepared from the anterior portion of the filariid. This amine induced concentration dependent increase in the membrane potential which possibly caused tonic paralysis of the filariid. The rate of micro filarial release by the female worms also declined in the presence of this amine. The study thus provided preliminary evidences for the presence of an octopamine neurotransmitter system and also about some of the roles it plays in A. viteae.
Subject(s)
Dipetalonema/physiology , Octopamine/physiology , Animals , Chromatography, Thin Layer , Dipetalonema/chemistry , Dipetalonema/growth & development , Electrophysiology , Female , Male , Membrane Potentials , Microfilariae/physiology , Movement/drug effects , Octopamine/biosynthesis , Octopamine/metabolism , Octopamine/pharmacology , Serotonin/pharmacologyABSTRACT
Parasitic filarial nematodes infect more than 200 million individuals worldwide, causing debilitating inflammatory diseases such as river blindness and lymphatic filariasis. Using a murine model for river blindness in which soluble extracts of filarial nematodes were injected into the corneal stroma, we demonstrated that the predominant inflammatory response in the cornea was due to species of endosymbiotic Wolbachia bacteria. In addition, the inflammatory response induced by these bacteria was dependent on expression of functional Toll-like receptor 4 (TLR4) on host cells.
Subject(s)
Drosophila Proteins , Onchocerca volvulus/microbiology , Onchocerciasis, Ocular/immunology , Onchocerciasis, Ocular/microbiology , Symbiosis , Wolbachia/pathogenicity , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Brugia malayi/physiology , Cornea/immunology , Cornea/metabolism , Cornea/microbiology , Cornea/parasitology , Dipetalonema/physiology , Doxycycline/pharmacology , Doxycycline/therapeutic use , Eosinophils/immunology , Humans , Immunity, Innate , Keratitis/immunology , Keratitis/microbiology , Keratitis/parasitology , Keratitis/pathology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Microscopy, Confocal , Neutrophil Infiltration , Neutrophils/immunology , Onchocerca volvulus/immunology , Onchocerca volvulus/physiology , Onchocerciasis, Ocular/parasitology , Onchocerciasis, Ocular/pathology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Toll-Like Receptor 4 , Toll-Like Receptors , Wolbachia/immunology , Wolbachia/physiologyABSTRACT
Although exogeneous "danger" signals such as LPS can activate APC to produce a Th1 response, the nature of events initiating a Th2 response is controversial. We now show that pathogen-derived products have the capacity to induce bone marrow-derived dendritic cell cultures to acquire a phenotype that promotes the differentiation of naive CD4+ T cells toward either a Th1 or Th2 phenotype. Thus, LPS-matured dendritic cells (DC1) promote a Th1 response (increased generation of IFN-gamma and reduced production of IL-4) by Ag-stimulated CD4+ T cells from the DO.11.10 transgenic mouse expressing a TCR specific for an OVA peptide (OVA323-339). In contrast, a phosphorylcholine-containing glycoprotein, ES-62, secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 Ab response in vivo, is found to induce the maturation of dendritic cells (DC2) with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-gamma). In addition, we show that the switch to either Th1 or Th2 responses is not effected by differential regulation through CD80 or CD86 and that a Th2 response is achieved in the presence of IL-12.
Subject(s)
Dendritic Cells/immunology , Dipetalonema/immunology , Helminth Proteins/immunology , Helminth Proteins/metabolism , Signal Transduction/immunology , Th2 Cells/immunology , Animals , Antigens, CD/physiology , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/physiology , B7-1 Antigen/physiology , B7-2 Antigen , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Differentiation/immunology , Dendritic Cells/cytology , Dendritic Cells/metabolism , Dipetalonema/physiology , Helminth Proteins/administration & dosage , Helminth Proteins/physiology , Immunoglobulin G/biosynthesis , Immunophenotyping , Injections, Subcutaneous , Interleukin-10/physiology , Interleukin-12/physiology , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Mice, Transgenic , T-Lymphocytes, Helper-Inducer/immunology , Th2 Cells/cytology , Th2 Cells/metabolism , Th2 Cells/parasitologyABSTRACT
Post-invasive third-stage larvae (pL3) of Acanthocheilonema viteae were labeled with [(35)S]-methionine in vivo, and proteins released into the culture supernatant before and during the third molt were analyzed. The molting supernatant (MSN) contained abundant proteins of 14, 18, 29, and 36 kDa. The 14- and 29-kDa proteins were exclusively found in the MSN, while the 18- and 36-kDa proteins were also produced by nonmolting pL3, albeit in much lower quantities. The cDNA for the most abundant protein in the MSN, an 18-kDa protein (Av18), was isolated by polymerase chain reaction (PCR) with reverse transcribed (RT) RNA of pL3, using information of the protein sequence. The Av18 full-length cDNA of 583 base pairs contained the 5' spliced leader sequence of nematodes, an open reading frame of 427 base pairs, and a poly(A) tail in typical distance to a polyadenylation signal. The deduced amino acid sequence encodes for a protein with a calculated size of 15.8 kDa. The N-terminus starts with a hydrophobic signal sequence and a predicted cleavage site after amino acid 20. The Av18 protein showed homologies to the deduced amino acid sequence of the larval transcripts Bm-alt-1 and alt-2 of Brugia malayi and to the Dirofilaria immitis proteins Di20/22 as well as to the Onchocerca volvulus proteins Ov-alt-1 and Ov-alt-2. Av18 is present in all parasite stages within the mammalian host, as determined by immunoblot with sera against the Escherichia coli-expressed protein and RT-PCR experiments. However, it was released into culture medium only by L3 and adult female worms. In female worms Av18 was localized in the cuticular region as demonstrated by immunofluorescent antibody tests using cryosections.
Subject(s)
Dipetalonema/physiology , Helminth Proteins/chemistry , Molting/physiology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Blotting, Western , DNA, Complementary/chemistry , DNA, Helminth/chemistry , Electrophoresis, Polyacrylamide Gel , Female , Fluorescent Antibody Technique, Indirect , Gerbillinae , Helminth Proteins/genetics , Male , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , RNA, Helminth/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , TicksABSTRACT
Evaluation of antifilarial activity of new potential agents in vivo is extremely time consuming and uneconomic. In the present study effort has been made to develop an in vitro screening method using Acanthocheilonema viteae, a subcutaneously dwelling rodent filariid with anaerobic metabolic characteristics like human filariids, W. Bancrofti/Brugia malayi as test parasite. Motility test and tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) based colorimetric assay were used as parameters in in vitro assay. Results showed that 92.3% of compounds (in vivo active) could be picked up in the in vitro assay when both adults and microfilarae (mf) were used simultaneously. Mf and adult stages separately detected, respectively, 84.6 and 69.2% of in vivo active compounds. The adults and mf separately and both the life stages together exhibited, respectively, 80.0, 50.0 and 80.0% false positive results in the in vitro test with in vivo inactive compounds. It is felt that mf stage when used in in vitro test using motility and MTT assays as parameters would be useful in primary screening of new potential filaricides.
Subject(s)
Dipetalonema/drug effects , Filaricides/pharmacology , Animals , Colorimetry , Coloring Agents/chemistry , Dipetalonema/growth & development , Dipetalonema/physiology , Dipetalonema Infections/drug therapy , Dipetalonema Infections/parasitology , False Negative Reactions , False Positive Reactions , Female , Male , Microfilariae/drug effects , Microfilariae/physiology , Movement/drug effects , Muridae , Oxidation-Reduction , Predictive Value of Tests , Tetrazolium Salts/chemistry , Thiazoles/chemistryABSTRACT
Filarial nematodes infect more than 100 million people in the tropics, causing elephantiasis, chronic skin lesions, and blindness. The parasites are long-lived as a consequence of being able to evade the host immune system, but an understanding of the molecular mechanisms underlying this evasion remains elusive. In this study, we demonstrate that ES-62 (2 microg/ml), a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B lymphocytes. Although this interaction is insufficient to cause B lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase and Ras mitogen-activating protein kinase pathways, and hence also to proliferation, via the Ag receptor. The active component of ES-62 appears to be PC, a molecule recently shown to act as an intracellular signal transducer, as the results obtained with ES-62 are broadly mimicked by PC alone. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES, by interfering with B cell function, could play a role in prolonging filarial infection in parasitized individuals.
Subject(s)
B-Lymphocytes/drug effects , Dipetalonema/physiology , Helminth Proteins/pharmacology , Lymphocyte Activation/drug effects , Phosphorylcholine/pharmacology , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein-Tyrosine Kinases/physiology , Rabbits , Sphingomyelin Phosphodiesterase/physiology , ras Proteins/metabolismABSTRACT
The quick and easy method of tetrazolium based colorimetric assay with MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to test the viability of the adult parasites of a rodent filariid Acanthocheilonema viteae in vitro. The ideal conditions required for antifilarial screening were determined by correlating the MTT reduction ability of worms with their size and age in the vertebrate host, also the duration of incubation and temperature of the in vitro culture. It was observed that the worms collected from the host after 90 days of L3 (infective larvae) exposure were not suitable for in vitro screen as they could not reduce MTT to that extent as the worms of early infection. Healthy and full grown worms and also those incubated at 37 degrees C for 16 hr or more caused maximum MTT reduction. Thus, it is recommended to select healthy adult filariids of proper age and size (male > 3.5 cm; female > 7.0 cm). The incubation temperature of the in vitro culture system needs to be adjusted to 37 degrees C and parasites might be exposed to drugs upto 24 hr without much alteration in MTT reduction of untreated controls.
Subject(s)
Colorimetry/methods , Dipetalonema/physiology , Filariasis/diagnosis , Muridae/parasitology , Tetrazolium Salts , Thiazoles , Animals , Cold Temperature , Coloring Agents , Female , Hot Temperature , MaleABSTRACT
The maintenance of the life cycle of Acanthocheilonema viteae is described with the aim to increase the production of parasite material using less experimental animals. The filaria was maintained in jirds (Meriones unguiculatus) and in soft ticks (Ornithodoros moubata). The optimal infection dosis for jirds was 80 infective larvae (L3). The mean worm number in groups of animals varied between 18 and 30 adult worms. A stable microfilaremia developed and only few animals developed pathological alterations as a consequence of the infection. A simple membrane feeding apparatus allowed mass feeding of ticks. Infection of ticks with microfilariae (mf) using this technique resulted in a mean no. of 594 +/- 527.2 L3/tick. L3 and mf were cryopreserved in liquid N2 with a simple technique. The described maintenance of the life cycle reduced the amount of required experimental animals to 30-40% of the originally needed numbers.
Subject(s)
Dipetalonema/physiology , Gerbillinae/parasitology , Ticks/parasitology , Animals , Dipetalonema/growth & development , Female , Male , Sex Ratio , Ticks/anatomy & histology , Time FactorsABSTRACT
After i.v. injection of 305 x 10(3) microfilariae (mf) per animal (50 g) into naive jirds, 50.8% of them could be recovered at autopsy 15 min later. Of these, 65.8% were calculated to be in the peripheral circulating blood (PCB) and were completely intact; 18.6% were recovered by perfusion of the lungs and 13.6% from the liver. In both organs about half the mf were associated with adherent lymphocytes and neutrophils but a few were partly disintegrated. Only 2.6% were recovered from the kidneys and the spleen. In long-term injection experiments using the same inoculum size the autopsy was done 15 min and 1, 3 and 6 weeks post-injection (p.i.) of mf into naive jirds. Throughout the experimental period the density of mf remained more or less constant in the PCB, but 3 weeks p.i. the density in the lungs increased up to 14 times to that in the PCB, whereas in the liver it decreased at the same time to a density similar to that in the PCB. In patent animals with adult worms delivering mf these were distributed as follows: 34.7% were calculated to be in the PCB; 24.4% were obtained by perfusion from the lungs and 22.0% from the liver; the rest were found in the kidneys (16.6%) and spleen (2.3%). In the lungs and the liver about 5/6 were associated with adherent cells, partly disintegrated or as fragments. In view of the fact that very few mf become disintegrated immediately after i.v. injection and also from their extremely long sojourn in the PCB, a low turnover rate of mf is presumed.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Dipetalonema Infections/veterinary , Dipetalonema/physiology , Gerbillinae/parasitology , Rodent Diseases/parasitology , Animals , Dipetalonema Infections/parasitology , Disease Models, Animal , Microfilariae/physiologyABSTRACT
Previous work has shown that the surface of infective larvae of parasitic nematodes will not bind the fluorescent lipid analogue 5-N-(octadecanoyl)aminofluorescein (AF18) until after exposure of the parasite to mammalian tissue-culture conditions. In this study, culture media which are permissive or non-permissive for the acquisition of lipophilicity for AF18 were altered in order to examine possible stimuli involved. This showed that external alkaline pH and high sodium ion concentration were highly stimulatory. The internal signalling pathways which may be involved in the surface alteration were then examined using agents which are known to affect intracellular signalling in mammalian cells. The results indicated that elevation of cGMP levels was stimulatory whereas inhibition of a putative Na+/H+ antiporter or calcium mobilization was inhibitory, and it is argued that high intracellular levels of cAMP may be inhibitory. Whilst the precise effects of the agents used on nematode cells remain to be established, these results provide a framework for the examination of the processes involved in the modification of the nematode surface which takes place immediately after the infection event.
Subject(s)
Nematoda/physiology , Nematoda/pathogenicity , Signal Transduction , 1-Methyl-3-isobutylxanthine/pharmacology , Aedes/parasitology , Animals , Brugia/drug effects , Brugia/pathogenicity , Brugia/physiology , Calcimycin/pharmacology , Cyclic AMP/metabolism , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Cyclic GMP/pharmacology , Dipetalonema/drug effects , Dipetalonema/pathogenicity , Dipetalonema/physiology , Gallic Acid/analogs & derivatives , Gallic Acid/pharmacology , Hydrogen-Ion Concentration , Larva , Mammals , Nicardipine/pharmacology , Nippostrongylus/drug effects , Nippostrongylus/pathogenicity , Nippostrongylus/physiology , Nitroprusside/pharmacology , Protein Kinase Inhibitors , Signal Transduction/drug effects , Sulfonamides/pharmacology , Trichinella/drug effects , Trichinella/pathogenicity , Trichinella/physiologyABSTRACT
The survival in culture of adult female Brugia pahangi, Acanthocheilonema viteae, and Onchocerca volvulus and adult male Onchocerca gibsoni was assessed by measuring parasite motility. Survival of all species was maximal in a nutritionally complex medium (RPMI-1640). All species survived for up to 48 hr in a simpler medium in which the only energy source was 10 mM glutamine; motility in this medium was dependent upon pH. For the species of Onchocerca, motility was maintained better in the presence of glutamine as the sole energy source than in glucose-only medium. Motility of B. pahangi incubated in 10 mM succinate was equivalent to that seen with 10 mM glutamine, but no other tricarboxylic acid intermediate supported this parasite in vitro. Antimycin A (1 microM) and potassium cyanide (KCN, 100 microM) paralyzed B. pahangi incubated in 10 mM glutamine, an effect antagonized by glucose. KCN at 10 or 100 microM was effective also against Onchocerca gutturosa in glutamine-only medium. Several glutamine antimetabolites reduced motility of B. pahangi by 72 hr. This inhibition was prevented by 2 mM glutamine. However, the inhibition of motility in the species of Onchocerca caused by these compounds was attenuated only partially by glutamine. These data demonstrate that, under certain conditions, filarial nematodes can utilize non-sugar substrates as energy sources. The differential sensitivity seen among these organisms to mitochondrial toxins and glutamine antimetabolites may be related to the extent to which they can use these alternative substrates to generate energy.
Subject(s)
Antimetabolites/pharmacology , Brugia pahangi/drug effects , Dipetalonema/drug effects , Glutamine/metabolism , Onchocerca/drug effects , Animals , Antimycin A/pharmacology , Brugia pahangi/physiology , Culture Media , Dipetalonema/physiology , Female , Glucose/metabolism , Glutamine/antagonists & inhibitors , Hydrogen-Ion Concentration , Isoxazoles/pharmacology , Male , Movement/drug effects , Onchocerca/physiology , Onchocerca volvulus/drug effects , Onchocerca volvulus/physiology , Potassium Cyanide/pharmacology , Sulfonamides/pharmacologyABSTRACT
The role of calcium in muscle contractility was explored in the filarial nematode Acanthocheilonema viteae (Dipetalonema viteae). The parasite was slit open longitudinally and mounted in a smooth-muscle chamber that had been filled with aerated (95% N2/5% CO2) physiological solution at 37 degrees C. Nifedipine (10(-6) M) and cadmium (3 x 10(-5) M) reduced the spontaneous isotonic contractions of A. viteae, whereas verapamil (10(-5) M) and diltiazem (10(-5) M) enhanced them. The effects of nifedipine and verapamil did not appear to be due to the solvent ethanol. All of the drugs reduced the maximal contraction induced by acetylcholine (ACh, 10(-5) M), although nifedipine was the most potent. After the exposure of worm preparations to a calcium-free medium containing ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 10(-4) M) for 1 h, application of ACh (10(-5) M) induced a small, transient contraction. Subsequent applications of ACh in this medium had no effect. Thus, the nematode muscle contraction appears to depend on extracellular calcium. Nifedipine, diltiazem, and verapamil could act by reducing the calcium influx across the muscle membrane.
Subject(s)
Calcium Channel Blockers/pharmacology , Calcium/physiology , Dipetalonema/drug effects , Acetylcholine/pharmacology , Animals , Cadmium/pharmacology , Diltiazem/pharmacology , Dipetalonema/physiology , Egtazic Acid/pharmacology , Female , Muscle Contraction/drug effects , Muscle Contraction/physiology , Nifedipine/pharmacology , Tetrodotoxin/pharmacology , Verapamil/pharmacologyABSTRACT
Exposure of A. viteae microfilariae to various lectins reduced their capacity to react with the peritoneal exudate cells of the host, Mastomys natalensis. Sugars corresponding to these lectins with the exception of N-acetyl glucosamine, did not affect the adhesion per se. They however, protected the parasite against the adverse effect of lectins. Neuraminidase and chitinase also suppressed adhesion capacity of the microfilariae. Except sodium dodecylsulphate which enhanced cell attachment, other surfactants inhibited this reaction considerably. The results indicate that antibody dependent adhesion of the microfilariae with the macrophages involves surface moieties of the parasite, where N-acetylglucosamine acts as the principal sugar residue. Participation of -SH groups also is inferred from the observations that p-chloromercuribenzoate and dithiobis-(2-nitrobenzoic acid) inhibited cell attachment and dithiothreitol provided protection against these agents.
Subject(s)
Cell Adhesion/physiology , Dipetalonema/physiology , Host-Parasite Interactions , Microfilariae/drug effects , Acetylgalactosamine/pharmacology , Acetylglucosamine/pharmacology , Animals , Dose-Response Relationship, Drug , Hexoses/pharmacology , Host-Parasite Interactions/drug effects , Hydrolases/pharmacology , Lectins , Muridae , Sulfhydryl Reagents/pharmacology , Surface-Active Agents/pharmacologyABSTRACT
The reproductive integrity and viability of adult female Acanthocheilonema viteae (syn. Dipetalonema viteae) maintained in culture for relatively long periods were assessed by transplantation into jirds. Worms cultured in chemically defined NI medium for approximately 3-4 wk remained active, but microfilarial release declined to barely detectable levels. Microfilarial production, however, was restored when the worms were transplanted subcutaneously into jirds. When cultured in NI medium beyond 4 wk no restoration of microfilarial production occurred on transplantation, presumably due to irreversible injury to the reproductive system. However, when NI medium was supplemented with fetal bovine serum resumption of microfilarial production occurred in transplanted females that had been in culture for as long as 2 1/2 mo. The addition of serum to NI medium played an important role in maintaining and protecting the functional integrity of the reproductive system.
Subject(s)
Dipetalonema Infections/parasitology , Dipetalonema/physiology , Animals , Dipetalonema Infections/blood , Female , Gerbillinae , Male , Microfilariae/physiology , ReproductionABSTRACT
1. Isotonic contractions were recorded from the filarial nematode, Dipetalonema viteae (Acanthocheilonema viteae), in an isolated tissue chamber. 2. Nicotine (10(-6) M) and pilocarpine (10(-5) M) increased the spontaneous contractions in the intact filariid, but acetylcholine (ACh, 10(-4) M) and muscarine (10(-5) M) were inactive. 3. When ACh was applied to an opened D. viteae, it was 10,000 times more potent. This indicates that the cuticle is an effective barrier to the penetration of ACh to the muscle cells. 4. The effects of ACh on the opened D. viteae were not affected by hexamethonium (10(-3) M) or atropine (10(-5) M) and were only partially reduced by (+)-tubocurarine (10(-4) M). 5. gamma-Aminobutyric acid (GABA, 10(-3) M) reduced the spontaneous activity of the intact D. viteae; however, the effect of GABA had a slow onset and recovery. Muscimol (10(-5) M) was more potent than GABA and had a more rapid onset and recovery. 6. GABA was 1,000 times more potent on the opened D. viteae than on the intact D. viteae. Baclofen (10(-3) M) was inactive on both preparations. 7. The effect of GABA was not antagonized by bicuculline (10(-4) M), picrotoxin (10(-5) M or penicillin G (10(-3) M). 8. It is concluded that the filariid cuticle acts like a lipid structure and blocks the penetration of polar substances, such as ACh and GABA. Also, due to the lack of efficacy of the ACh and GABA antagonists, it was concluded that the nematode receptors are somewhat different from the mammalian ACh and GABA receptors.
Subject(s)
Acetylcholine/pharmacology , Dipetalonema/physiology , gamma-Aminobutyric Acid/pharmacology , Action Potentials/drug effects , Animals , Cholinergic Antagonists , Cricetinae , GABA-A Receptor Antagonists , Isotonic Contraction , Muscle Contraction/drug effects , Muscles/physiology , Receptors, Cholinergic/drug effects , Receptors, GABA-A/drug effectsABSTRACT
Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.
Subject(s)
Azo Compounds/metabolism , Dipetalonema/physiology , Formazans/metabolism , Tetrazolium Salts/metabolism , Animals , Colorimetry , Female , Indicators and Reagents , Oxidation-ReductionABSTRACT
Studies were conducted on survival and microfilarial release of afult Dipetalonema viteae in culture, using worms of various ages derived from jirds. In chemically defined NI medium (a 1:1 mixture of NCTC 135 and Iscove's modified Dulbecco's medium) under a gas phase of 5% CO2 in nitrogen (pO2 of medium approximately 40 mm Hg), the peak of microfilarial release of several thougsand microfilariae per female per 24 hr occurred at approximately day 10. Thereafter, microfilarial release declined and generally ended about 1 mo after the start of culture. The adult females moved actively for about 50 days or more and survived up to 82 days in NI medium alone. The females in NI medium supplemented with fetal bovine serum showed serpentine movement for approximately 2 mo. Some of the worms survived more than 83 days. The total number of microfilariae deposited in culture by D. viteae increased as adult females grew in size (volume) over time. Microfilarial deposition continued to increase after worms reached maximum size, deposition reaching a plateau between approximately 300 and 400 days of age. Thereafter, microfilarial deposition decreased as females continued to age. Addition of fetal bovine serum to the NI medium increased the number of microfilariae released and extended the period of release.
Subject(s)
Dipetalonema/physiology , Animals , Blood , Culture Media , Dipetalonema/growth & development , Female , Microfilariae/physiology , MovementABSTRACT
Fourteen synthetic retinoids with known and different binding affinities to retinol binding proteins of Dirofilaria immitis, retinol, and retinoic acid were tested in vitro against female Litomosoides carinii (drug levels 20, 10, 1 nM/ml) and against microfilariae of L. carinii, Brugia malayi, B. pahangi and Acanthocheilonema viteae (drug levels 100, 20, 10, 1 nM/ml). All compounds including retinol and retinoic acid had at least some effects on the filarial parasites. Except for 3 synthetic retinoids, continuous exposure of adult L. carinii to the drugs reduced the motility of the worms completely or remarkably by day 7 of incubation in a dose and time dependent fashion. Also, the release of microfilariae was completely or remarkably suppressed in a dose and time dependent manner by 20 and 10 nM/ml of all except 4 of the retinoids. Short term exposure to the drugs (up to 20 nM/ml) for 4 h followed by subsequent incubation in drug-free medium was ineffective except for one synthetic retinoid (13-cis-N-(2-hydroxyethyl)retinamide:13-cis-Her). Effects on microfilariae were also dose and time dependent. All compounds affected markedly the motility of L. carinii microfilariae within 20 h at dose levels of 1 nM/ml and above. Microfilariae of B. malayi, B. pahangi and especially of A. viteae were generally less sensitive. Eight of the synthetic retinoids, but not retinol and retinoic acid, were effective (10 nM/ml). There were generally no correlations between the various effects of individual compounds; i.e., activities varied within one species depending on the parameters used and depending on the parasite species.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Anthelmintics/pharmacology , Filaricides/pharmacology , Filarioidea/drug effects , Retinoids/pharmacology , Animals , Brugia/drug effects , Brugia/physiology , Dipetalonema/drug effects , Dipetalonema/physiology , Female , Filarioidea/physiology , Microfilariae/drug effects , Movement/drug effects , Tretinoin/pharmacology , Vitamin A/pharmacologyABSTRACT
Previous attempts to assess nematode viability have been critically reviewed and the need to apply more objective biochemical criteria emphasized. The practicalities of assay development have been discussed with regard to sensitivity, selectivity and methodological considerations. The biochemical basis and assay methology for six assays (adenine leakage, adenine uptake, leucine uptake, 14CO2 evolution, lactate output and MTT reduction) that we have recently evaluated are detailed. The viability of Acanthocheilonema viteae females exposed for 120 h in vitro to 17 standard compounds (at 10 microM) has been assessed using these six assays and compared relative to motility indices from the micromotility meter. It was concluded that, despite the slightly superior sensitivity of the 14CO2 evolution assay, the MTT reduction method was most suitable for field use due to its technical and practical simplicity, and its applicability to fragments of onchocercal tissue. It was suggested that, in the absence of a better in vitro assay, the feasability of using MTT reduction together with histology should be assessed in a validation exercise with Onchocerca gibsoni.