Molinema Dessetae Research Articles

Odile Bain (April 28, 1939–October 16, 2012): A Life Dedicated to Systematics and Biology of Filariae

​OdileOdile Bain was born on April 28, 1939, in Dalat, Vietnam, where her father, a military officer, was based. She went to high school in Dakar, Senegal, and graduated in biology in Rennes (France) in 1960. She taught biology at a high school for two years and then worked as a teaching assistant at the Faculty of Rennes in 1962. In 1963, she started a postgraduate course in histology in Paris. Odile moved to the Natural History Museum in Paris, France, in September 1964 to join the Helminth Zoology laboratory of Prof. Alain Chabaud. It was there that she began to build an incredible career in parasitology. She first graduated with her PhD (“these de 3eme cycle”) in histology in April 1968. Then, in April 1971, she was awarded her “Doctorat d'Etat es Sciences Naturelles” for her work on “Evolution des filaires chez le vecteur: morphologie larvaire et mecanismes du passage des microfilaires dans l'hemocele.” In the meantime, she was appointed by the CNRS (Centre National de la Recherche Scientifique) in October 1965 as an undergraduate research assistant; then she was granted a position as a CNRS research associate (chargee de recherche) in October 1971 and, later, in January 1984, as a CNRS research supervisor (directeur de recherche). Image 1 Photo courtesy of Kerstin Junker (Ile de France, 2008). The laboratory is a parasitology history lesson in itself. It was founded in 1961 by Prof. Chabaud and gathered specialists of different groups of “worms” and protozoa. Prof. Chabaud supervised several teams of research on parasitic nematodes, trematodes, cestodes, and protozoa. The laboratory was originally located on rue Cuvier but moved to its present location on rue Buffon in 1981. The laboratory quickly established itself as a centre of excellence, but it retained an unbridled hospitality supported by good food and a sucession of resident canine friends. It is in this laboratory that Odile honed her parasitological and morphological skills. She had a passion for biology and microscopy that was unrivalled and universally recognised. In her 50-year career, she published more than 360 articles (Text S1) and had 30 years of successive contracts with the World Health Organization (WHO), the Edna McConnell Clark Foundation, and the European Union. In 1974, she received the bronze medal of the CNRS, and in 1984, she won the Foulon prize from the French Academy of Sciences for her work on the zoology and parasitology of filarial infections. She was also the curator of the zooparasitic nematodes collection at the French National Natural History Museum, Paris. Odile's primary expertise was the systematics of filariae, and her intimate knowledge of the biology of filariae led to characterization of the phyletic relationships between filarial lineages and those of non-filarial nematodes. Odile's group studied a large number of filarial species collected in many countries, which led to countless worldwide collaborations. Over time, the scope of her research expanded to encompass the medically relevant filarial species that cause diseases of humans in the tropics. In these studies she investigated aspects of vector biology that relate to filarial worm infection with the end goal of devising a means to control transmission. She also developed a strong interest in finding and identifying the vectors of lesser-known zoonotic filarial species. Her knowledge of animal filariae also resulted in the establishment of experimental models of filariasis in rodents (Monanema martini with skin-dwelling microfilariae, Molinema dessetae), including Litomosoides sigmodontis, which is the only filaria capable of establishing patent infections in laboratory mice. Use of these models to study host–parasite interactions has significantly advanced the study of filarial disease, chemotherapy, and immunology. Besides being a most highly respected scientist who strove for only the highest quality research, Odile Bain was an eternal and energetic optimist who welcomed everyone with friendliness, and whose enthusiasm and curiosity were infectious. We all deeply feel her loss and will remember the smiling Odile, always ready to take on new challenges in science.

In-vitro evaluation of filaricidal activity of GABA and 1,3-dipalmitoyl-2-(4-aminobutyryl)glycerol HCl: a diglyceride prodrug

A diglyceride ester of gamma-aminobutyric acid (GABA) has been synthesized and its filaricidal activity compared with GABA, and progabide in-vitro, on infective larvae and microfilariae of Molinema dessetae, a rodent filaria. GABA induced paralysis in infective larvae but was inactive on microfilariae. There were interactions between the culture medium and GABA. The ester drug at 0.1 mmol L-1 (1,3-dipalmitoyl-2-(4-aminobutyryl)glycerol HCl) was as active as progabide on infective larvae and hundredfold more potent than GABA. Its microfilaricidal activity at 1 mmol L-1 was lower than that progabide at 0.1 mmol L-1 but a delayed effect was observed. The data confirm filariae sensitivity to GABA derivatives.

Since phenothiazines alter antibiotic susceptibility of microorganisms by inhibiting efflux pumps, are these agents useful for evaluating similar pumps in phenothiazine-sensitive parasites?

Phenothiazines have activity against Schistosoma mansoni, Trypanosoma brucei, Trypanasoma gambiensi, Molinema dessetae, Leishmania spp., Plasmodium falciparum and free-living protozoa. These organisms and other parasitic infections are prevalent in HIV-infected humans. These infections are becoming more frequently resistant to commonly employed antibiotics, and due to the absence of economic motivation, new and effective compounds against these infections are not anticipated in the near future. Resistance of prokaryotes and eukaryotes to antibiotics is now known to be also due to the presence of efflux pumps that extrude the antibiotic prior to the agent reaching its target. Because phenothiazines are known to inhibit some efflux pumps and therefore alter the susceptibility of the organism to an antibiotic to which it is resistant, and also because of the sensitivity of the above parasites to phenothiazines, efflux pumps may play a role in emerging antibiotic resistance of these organisms. Furthermore, their prevalence is known to be greatest in areas that have high rates of HIV infection; therefore, it would be necessary that these agents should receive close scrutiny. This review concerns the attributes afforded by phenothiazines related to their effective activity against a wide range of parasites. Because these agents are inexpensive and many are no longer protected by patent, they may be exploited as anti-parasitic agents in the poorer areas of the world.

Research note In vitro antifilarial activity of organometallic complexes against infective larvae of Molinema dessetae and adult females of Brugia pahangi

New organometallic complexes having protozoocidal properties were evaluated for their in vitro antifilarial activity using two models: infective larvae of Molinema dessetae and adult females of Brugia pahangi. The compound most active on the M. dessetae model was Ir(I)-COD-pentamidine tetraphenylborate with an EC 50 = 6±1 μM after 7-day-incubation. In the 2-aminobenzothiazole series, Ruthenium was more potent than Iridium for antifilarial activity. A dithiocarbamate function significantly enhanced the antifilarial activity. The compounds derived from benzimidazole were inactive whatever the metal (Iridium or Rhodium). The other compounds exhibited EC 50 ranging from 10 to 31 μM. On adult female Brugia pahangi in vitro, Pt-DDH-N-acetylleucine, Pt-diminazene and Pd-Cl 4-piperazine at 20 μM began to kill both microfilariae and the developing embryos within the mothers on day 2. The compounds, except for Pd-Cl 4-piperazine, killed the adults after 5 days. Rh-Cl-2-chloropyridine caused obvious slowing of the adults from day 3 onward but did not affect the viability of adults, microfilariae or developing embryos. In vivo antifilarial investigations are necessary to appreciate the real advantage of heavy metal complexes in the experimental treatment of filariasis.

Lymphotropic antifilarial agents derived from closantel and chlorambucil

New closantel and chlorambucil prodrugs expected to accumulate in the lymphatic system were evaluated on the filaria Molinema dessetae. The prodrugs of closantel had a delayed effect in vitro on the infective larvae compared to the free drug. The closantel prodrugs were less toxic in vivo than closantel itself. The most active prodrug after treatment at 200 μmol/kg by the oral route was the 1,3-dipalmitoyl-2-succinyl-glycerol-closantel. The macrofilaricidal delayed effect of closantel prodrugs was of interest to prevent anaphylactic shock. In vitro, chlorambucil was active on M. dessetae infective larvae with an IC 50 of 26 μM. 1,3-Dipalmitoyl-2-chlorambucil-glycerol was slightly active while the addition of a thioether function between the drug and the lymphotropic ligand canceled the activity. However, no activity with chlorambucil and its prodrugs was observed in vivo. The lymphotropism of these prodrugs has now to be verified using comparative pharmacokinetics in serum and lymph to quantify the increase in drug concentration in lymph.

Purification and characterization of lactate dehydrogenase isoenzymes 1 and 2 from Molinema dessetae (Nematoda: Filarioidea)

High levels of lactate dehydrogenase (LDH; EC 1. 1. 1. 27) activity have been detected in the filarial worm Molinema dessetae. The two major LDH isoenzymes (LDH1 and LDH2) from female worms were purified by successive chromatography on diethylaminoethyl (DEAE)-Sepharose, carboxymethyl (CM)-Sepharose, and hydroxyapatite columns followed by fast protein liquid chromatography (FPLC)-gel filtration. LDH1 and LDH2 isoenzymes were found to be dimers with subunits of 58 kDa. They had similar properties with regard to substrate and coenzyme affinity. The apparent Michaelis constants (K(m) values; mean +/- SEM, n = 10) were 0.34 +/- 0.04 mM for pyruvate, 0.25 +/- 0.02 mM for reduced nicotinamide adenine dinucleotide (NADH), 2.5 +/- 0.21 mM for lactate, and 0.18 +/- 0.02 mM for NAD, which suggested that pyruvate reduction was the favored reaction. LDH1 and LDH2 were affected by p-chloromercuribenzoate and Hg2+, and such inhibitory effects could be reversed by the addition of thiol compounds (L-cysteine or beta-mercaptoethanol) as observed for mammalian LDH. Oxalate acted as a noncompetitive inhibitor of pyruvate reduction (Ki = 4.7 +/- 0.35 mM; mean +/- SEM, n = 10) and as a competitive inhibitor with lactate (Ki = 2.3 +/- 0.21 mM), whereas oxamate acted as a competitive inhibitor with pyruvate (Ki = 3.3 +/- 0.28 mM) and was noncompetitive with lactate (Ki = 19 +/- 1.2 mM). These substrate analogues exerted similar effects on mammalian LDH, but the inhibition constants were significantly different. The existence of structural and kinetic differences between mammal and filarial LDH isoenzymes prompted us to evaluate them as targets for chemotherapeutic attack.

Synthesis and nematocidal activities of new analogs of pyrantel

Summary A set of new analogs of pyrantel was synthesized in good yields by lithiation of 1,2-dimethyltetrahydropyrimidine with n -butyllithium in tetrahydrofuran and condensation with aromatic esters. Spectrometric studies showed the large influence of intramolecular bonding in the tautomeric equilibria between the possible structures. Anthelmintic screening showed in vitro efficiency against Molinema dessetae , but a weak activity against Rhabditis pseudoelongata and Nippostrongylus brasiliensis .

Synthesis, antifungal and nematocidal activities of thioureines with an aminoester sequence

Twenty-three arylthioureines bearing β-alanine or γ-aminobutyric alkyl ester chains were synthesized for in vitro screening toward 44 strains of fungi and 2 genera of nematodes. The nitro derivatives were the most potent compounds against Aspergillus and Candida strains. Ester chains increase activity against the filarial worm Molinema dessetae. Twelve compounds have EC 50 < 40 μg/ml. However, the anthelmintic potency is weak compared with tetramisole

Isoenzymatic diagnosis of filariae: a method for separation of lactate dehydrogenase isoenzymes from Molinema dessetae (Nematoda: Filarioidea)

Lactate dehydrogenase (LDH) is highly active in filariae and could be a valuable tool for phyllogeny studies. Unfortunately, the isoenzymatic diagnosis of filariae is often difficult for LDH because of a poor mobility of the enzymes in starch gels which are the most commonly used in such studies. We propose here a method to separate filarial LDH isoenzymes using disc electrophoresis. The experiments were carried out on male and female Molinema dessetae in order to compare their respective isoenzymes. The study of several parameters such as buffer systems, percentage of bisacrylamide and progression time led to optimize the enzyme separation. LDH from male and female filariae were compared to mammal LDH-H4 and LDH-M4. Five and four LDH isoenzymes were found, respectively, in male and female worms. Relative concentration of each isoenzyme diverged between male and female worms. Mammal muscle LDH-M4 type moved between LDH2 and LDH3 from female worms, and between LDH1 and LDH2 from male worms. Mammal heart H4 type enzyme was very different in electrophoretic mobility. The ratio of each isoenzyme was determined by densitometry. The major isoenzymes from female worms will be studied as a biochemical target for chemotherapeutic attack.

Larval biology of six filariae of the sub-family Onchocercinae in a vertebrate host.

The development of six filariae of the sub-family Onchocercinae-Litomosoides sigmodontis, Acanthocheilonema viteae, Molinema dessetae, Monanema martini, Brugia malayi, B. pahangi-was compared in rodents, following a single inoculation of a low or high dose of infective larvae. Analysis was done with 105 rodents dissected and 53 rodents fixed for histopathology. The percentage of larvae which developed corresponded to the proportion of those which were able to penetrate into the sub-cutaneous lymphatic vessels; this percentage was determined during the first day (phase 1) and was characteristic of the filaria-host pair, and independent of the number of larvae inoculated. It could remain stable for a long time, more than eight months with M. martini (phase 2); the phenomena of regulation appeared later (phase 3). The larvae migrated through the lymphatic system, which represents a medium less protected and thus less aggressive than the blood system. The coelomic cavities, almost devoid of inflammatory cells, represented an ultimate shelter, as well as the joint-cavities (colonized by some Dirofilariinae). Localizations in the cardio-pulmonary blood system were accidental and occurred when, during the migrations, some larvae penetrated into the thoracic channel and arrived in the superior vena cava, then the right ventricle and the pulmonary arteries (the biology of Dirofilaria immitis resulted in a secondary adaptation); such accidents may occur with adult filariae, especially, after drug treatment. One may expect similar events in human filariasis. These "occult" filariae, more frequent than it is usually thought, influence the immunological status and the pathology.

Study of lymphotropic targeting and macrofilaricidal activity of a melphalan prodrug on the Molinema dessetae model.

This study deals with the design of a new macrofilaricidal drug derived from melphalan and having a lymphotropism to avoid the hepatic first pass effect and enhance bioavailability after oral administration. Melphalan was linked to a ligand leading to a prodrug called 1,3-dp-melphalan which has structural analogy to triglycerides. The Molinema dessetae/Proechimys oris model was used for antiparasitic evaluation. Melphalan was macrofilaricidal in vitro against Molinema dessetae at 1mM, inactive in vivo after an oral single dose at 164 mumol/kg while the prodrug 1,3-dp-melphalan was active against adult worms after a single dose at 82 mumol/kg. After an oral administration of the prodrug to rats, the maximum concentration and the cumulated quantities of melphalan in lymph were about 45-fold higher than those observed with the free drug under the same conditions. Moreover, the plasma concentration of melphalan was 2-fold higher than those observed after the administration of the free drug. These results are in favor of lymphotropic targeting as a novel approach to develop new orally active macrofilaricides.

Research for new filaricides by using phosphoenolpyruvate-carboxylase from Molinema dessetae (Nematoda: Filarioidea) as biochemical target

Loiseau P. M., Marchat L., Depreux P., Gayral P. and Petek F. 1993. Research for new filaricides by using phosphoenolpyruvate-carboxylase from Molinema dessetae (Nematoda: Filarioidea) as biochemical target. International Journal for Parasitology 23: 697–698. In the aim to search for new antifilarial drugs, we propose here a study of phosphoenolpyruvate-carboxylase (E.C.4.1.1.31) as a therapeutic target. Among some potential inhibitors, compounds Nos. 5 and 9 which are phenoxycyclohexane derivatives were inhibitory with K i values respectively at 122 and 93 μ m. The inhibition was found to be noncompetitive. Concerning compound No. 5, the drug concentrations responsible for an enzyme inhibition of 50% were about 2-fold higher than those necessary to kill the adult parasite in vitro. Such results suggest the existence of another mechanism involved in this drug's action. Nevertheless, the correlation between in vitro results on adults and enzyme assay appeared clear and validates PEP-carboxylase as a biochemical target which should be investigated using a series of substrate analogues.

Synthèse, activités anti-parasitaire et anti-fongique d'arylalkyl- et d'arylvinylthiazolines

Twenty-seven arylalkyl- or arylvinylthiazolines were synthesized and tested in vitro against three genera of nematodes, various yeasts and opportunistic fungi. Vinyl compounds seem to have an interesting filaricidal activity against Molinema dessetae and anti-fungal activity against yeasts.

In vitro antifilarial evaluation of phenoxycyclohexane derivatives.

Numerous potential inhibitors of the phosphoenolpyruvate-carboxylase found in Molinema dessetae were synthesized and evaluated as inhibitors of the purified enzyme and as killers, in vitro, of M. dessetae adult females and infective larvae. Phosphoenolpyruvate analogs (or derivatives) appeared disappointing whereas phenoxycyclohexane derivatives inhibited the enzyme in a non-competitive manner and killed the parasites. The compounds P2281 and P2285, for example, had Ki values of 122 and 93 microM, respectively. In vivo tests of the most effective phenoxycyclohexanes will now be carried out to check their potential as antifilarial drugs.

Purification and properties of phosphoenolpyruvate carboxylase from Molinema dessetae (Nematoda: Filarioidea)

The presence of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), an enzyme at the branchpoint of glycolysis and the Krebs cycle was detected in the Filaria Molinema dessetae. This enzyme has not previously been identified in Helminths, which have so far been found to only possess a phosphoenolpyruvate carboxykinase (EC 4.1.1.32). This enzyme had a level of activity comparable to that of pyruvate kinase, and was relatively less active than enzymes such as malate dehydrogenase or lactate dehydrogenase. We propose here a method of purification of M. dessetae PEP-carboxylase. When purified to electrophoretic homogeneity, the enzyme had a molecular weight of 64 kDa. Kinetic studies indicated that the carboxylation reaction had an optimal pH of 5.8. The enzyme was inhibited by cations such as Fe2+, Zn2+, Cd2+, Cu2+ but required the presence of Mg2+ or Mn2+. The enzyme was thermostable. The apparent Km value of 2.38 mmol for phosphoenolpyruvate for the carboxylation reaction was higher than previously reported values. The Km value for KHCO3 was found to be 1.6 mmol. PEP-carboxylase did not catalyse the reverse reaction.

Une nouvelle classe de composés anti-parasitaires: les spiroarsoranes. Evaluation in vitro sur la filaire Molinema dessetae

A new class of biologically active compounds ‘spiroarsorans’ was synthesized and their filaricidal activity tested in vitro against Molinema dessetae. Products showed an important activity associated with low toxicity.

Nouvelles données sur la lignée Dipetalonema (Filarioidea, Nematoda)

The evolutionary line of Dipetalonema can apparently be divided into four groups: I: Australian species; II: paleoendemic South American species; III: the Tetrapetalonema group; IV: the Acanthocheilonema group. Loxodontofilaria at present insufficiently known to be classified and several species belonging to the Acanthocheilonema group are the object of the present study. Descriptions are given of Loxodontofilaria asiatica n. sp., parasite of Elephas indicus in Burma, Cercopithifilaria degraaffi n. sp., parasites of Papio ursinus in South Africa, C. cephalophi n. sp., parasite of Cephalophus dorsalis and C. gabonensis n. sp., parasite of Atherurus africanus in Gabon. Additional morphological data are given on Cercopithifilaria didelphis, C. rugosicauda, Acanthocheilonema pachycephalum, A. viteae, Molinema dessetae, Dipetalonema gracile, Orihelia sp., Skrjabinofilaria skrjabini, Breinlia (B.) spratti, Litomosa sp., Loxodontofilaria hippopotami. Yatesia n. gen. with type species Yatesia hydrochoerus (Yates, 1980), is proposed, distinguished by specialized characters of the posterior extremity. The genus Cercopithifilaria is used to accomodate species considered as specialized Acanthocheilonema. Chenofilaria is placed in synonymy with Acanthocheilonema. Loxodontofilaria includes the three filarid species from elephants, L. loxodontis, L. gossi, L. asiatica n. sp. and the species from the Hippopotamus, L. hippopotami; D. okapiae is considered a species inquirenda. The interpretation given for the neotropical fauna is the following: --Skrjabinofilaria, Orihelia, Dasypafilaria and Dipetalonema may be true paleoendemics in South America. --Molinema and Ackertia on the one hand and Yatesia on the other may be forms of African origin introduced at the end of the Eocene during the migration of African rodents into South America. The cpature in American reptiles (the genus Macdonaldius) could have occurred during this period. --Surprisingly, the two species of Dipetalonema in Didelphis may be late captures of neartic origin: A. pricei from Acanthocheilonema in carnivores and C. didelphis from a Cercopithifilaria in eutherian mammals.