Chemotherapy for onchocerciasis: results of in vitro experiments with promising new compounds.

An improved short-term in vitro culture system was used for the routine screening of hundreds of promising new compounds with the target organism, the filarial nematode Onchocerca volvulus. The most active leads were identified among the pyrimidinylguanidines, amidine derivatives, the imidazolinylhydrazones, thiosemicarbazone derivatives and thiadiazole derivatives. Single compounds of these leads demonstrated strong macrofilaricidal efficacy in minimum effective dose trials down to 0.1 microM and in experiments evaluating the minimum time of exposure after less than 6 h exposure. In the group of the pyrimidinylguanidines we found a significant correlation of structure and activity: change of a single side-group in the molecules had dramatic influence on compound activity. Most of the new compounds that were active on the macrofilariae did not show significant activity on microfilariae (mf) in in vitro trials. Only one compound with significant activity against female O. volvulus worms killed mf at very low concentrations. Some of the promising leads will be processed in further trials on a preclinical level with predictive cattle models.

Demonstration of immunoglobulin G antibodies against Onchocerca volvulus excretory-secretory antigens in different forms and stages of onchocerciasis.

The excretory-secretory (E-S) products of helminths are considered to comprise immunogenic molecules of high diagnostic value. In the present study, the serodiagnostic potential of the E-S products released in vitro by cultured female Onchocerca volvulus was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting using 190 serum samples from persons infected with O. volvulus and unexposed persons. The sensitivity of detection of anti-O. volvulus E-S antibodies was 94% for sera from patients with the generalized form of onchocerciasis and 100% for sera from patients with the chronic hyperreactive form (sowda). 95% of the sera from amicrofilaridermic persons, who subsequently became microfilaridermic within 2 years, reacted with O. volvulus E-S antigens and the donors were therefore regarded as having had a prepatent infection when first examined. These sera gave higher (P < 0.05) ELISA optical densities than sera from the same persons obtained when they had become patent, indicating a loss of antibody reactivity after emergence of microfilariae. The specificity of the E-S ELISA was 100% when sera of subjects infected with Wuchereria bancrofti were used, and at least 88% for Mansonella perstans sera. In Western blot analysis, the sera of persons with generalized onchocerciasis recognized 7 protein bands. Many E-S proteins were stained less intensely by the sera of subjects with generalized onchocerciasis than by the sera of sowda patients. Similar antigen bands were demonstrated using sera from the persons with prepatent infections.

The anterior central nervous system of male Onchocerca volvulus (Nematoda:Filarioidea) as a target for chemotherapy: results of an electron microscopy study.

An electron microscopy study was performed to evaluate the feasibility of the use of the anterior nerve ring of male Onchocerca volvulus for the assessment of early drug effects. Worms were exposed to new and known compounds at reasonable concentrations of 1 microM and less for 6, 12, 18, and 36 h in an established in vitro system. The anterior end of the filariae up to a length of 1 mm was examined and the morphological findings were compared with motility and reduction of a tetrazolium sat to formazan by live but not dead worms. The nerve fibers were more susceptible to the chemotherapeutic intervention then the other tissues in the anteriormost part of the worms. The alterations depended on the duration of exposure and the chemical nature of the compounds used. Morphological changes in the nervous tissue and the inhibition of motility and formazan production corresponded well for the arsenical mel w, used as an active standard, two pyrimidinyl-guanidines (PD 105482 and PD 105666), and an imidazolinylhydrazone (WR 251993).

The ultrastructure of the anterior end of male Onchocerca volvulus: papillae, amphids, nerve ring and first indication of an excretory system in the adult filarial worm.

A detailed morphological investigation of the anterior sensory organs, the nerve ring and a glomerulus-like structure in male Onchocerca volvulus was performed by means of electron microscopy. The 8 head papillae are arranged in the common 4 + 4 pattern of most filarial worms in circles around the mouth opening. The amphidial openings are found between the circles of inner and outer papillae on both sides of the mouth. Inside, several additional nerve axons are seen in the tissue of the anterior tip not related to one of the identified papillar structures. The inner and outer papillae exhibit a remarkably different fine structure, and are part of a complex system of at least 2 different receptor cell types at the anterior tip of the worm. The amphidial channel contains 8 modified cilia; accessory axons are associated with the cytoplasm of the sheath cell. The anterior nerve ring of male worms is located about 150 micrometers posterior from the outermost tip of the head region. It consists of several fibres coiled around the oesophagus. The comparison of the fine structure of the central nervous system did not show the expected morphological differences associated with the heterogeneous age distribution in the natural worm population. This was in contrast to previous findings with respect to tissues in different parts of the worm. The study also provides the first evidence that suggests the existence of an excretory organ in a filarial worm in the region of the anterior nerve ring. Paired glomerulus-like structures in the lateral chords and a canal formed by a projection of the basal zone of the cuticles were identified.

Ultrastructure study of the excretory system and the genital primordium of the infective stage of Onchocerca volvulus (Nematoda:Filarioidea).

The electron microscopic investigation of the anterior part of the infective third-stage juvenile of Onchocerca volvulus provides first insights into the structure of the excretory system of this developmental stage of the parasite. The most anterior part of this system consists of a cell process of the syncytial excretory cells. At this height the excretory cells enclose the cuticle-lined excretory channel. The channel is in the process of elongation in the anterior-posterior direction, indicated by cell division in this region. More posteriad an ampulla-like structure is forming in the cytoplasm of the excretory cells. The inner surface of this ampulla is lined with a small number of single microvilli. In this part of the system the cytoplasm of the excretory cells is rich in Golgi bodies and endocytic vesicles. The ampulla has direct access to the exterior by the excretory duct. The excretory duct is a cuticle-lined structure surrounded by supporting fibres of an additional cell. This duct cell connects the excretory duct to the body-wall cuticle at the excretory pore. Adjacent to the region of the excretory system a cell is found that resembles a gland cell. This cell is in close contact to the ventral nerve cord. The genital primordia of the third-stage juvenile consist of several dividing cells. The female genital primordium is seen at the junction of the muscular with the glandular oesophagus and the male primordium can be found at the junction of the glandular oesophagus with the gut.

Viability of Onchocerca volvulus in vitro.

The use of a selective schedule of tests to identify a viable population of isolated adult Onchocerca volvulus (Nematoda: Filarioidea) has been investigated in a large worm population. The study was initiated to develop methodology appropriate to test new candidate macrofilaricides for their in vitro activity against O. volvulus. After removal from the host the viability of isolated intact parasites was estimated by assessing the motility indices of male worms, and the colorimetric quantification of the reduction of the bioreducible tetrazolium reagent XTT and lactate output by female worms. Additionally the motility of whole females and the movement of inner organs of female worms were scored quantitatively. These response parameters were used to sort the adult worms into viability groups at the start of the in vitro culture. The adult worms were then observed for 6 days and viability was assessed regularly during the culture period. At the end of the culture period, the reduction of the water-insoluble tetrazolium reagent MTT was used to determine the formazan formed by the entire male and female worms. The response parameters used at the start of the culture proved to be highly predictive for detecting viable and non-viable adult worms. In the group of worms selected as 'viable' around 70% kept their motility and metabolic activity at a high level until the end of the culture compared to the initial level. In contrast, none of the female worms and only 13% of the male worms categorized as 'poorly viable' demonstrated a motility index or metabolic level at the end of the culture period that was comparable to that of the worms in the 'viable' groups. For female worms the lactate output correlated significantly with weight whereas no correlation was seen between MTT reduction and weight.

Characterization of a recombinant T cell and B cell reactive polypeptide of Onchocerca volvulus.

To identify potentially protective Ag of the filarial nematode Onchocerca volvulus on the molecular level we screened a cDNA library of O. volvulus with a human serum raised against radiation-attenuated infective larvae of O. volvulus. A cDNA clone of 218 bp (OvL3-1) was selected for further studies. It was expressed in Escherichia coli and affinity purified recombinant polypeptide was tested for its ability to stimulate in vitro PBMC from African onchocerciasis patients and PBMC from chimpanzees experimentally infected with O. volvulus. An enhanced cell proliferation by PBMC was observed in many patients after stimulation with the recombinant OvL3-1 polypeptide. In addition, some patients' PBMC responded to OvL3-1 stimulation with enhanced IL-2 production. Infected chimpanzees also showed an increase in T cell proliferation. Onchocerciasis patients had variable levels of specific antibodies directed to the recombinant polypeptide when sera were tested by ELISA. A mAb directed against the recombinant protein located the native target Ag in the muscles of the adult worm. The molecular mass of native OvL3-1 was found to be 50 kDa on immunoblots. Polymerase chain reaction analysis of RNA from different life stages of the parasite showed that OvL3-1 is transcribed in all parasite stages within the mammalian host. A homologous gene is also present in other filarial parasites. The protein corresponding to OvL3-1, therefore, represents an immunogen present during the whole life-span of the parasite, and because of its B and T cell stimulatory properties, it may be a candidate for a protective Ag in human filariasis.

Ultrastructural observations on the nervous system and the sensory organs of the infective stage (L3) of Onchocerca volvulus (Nematoda: Filarioidea).

A detailed morphological investigation of the sensory organs and the nervous system of the third juvenile stage of Onchocerca volvulus was performed at the ultrastructural level. A complex system of different receptor cells is found at the anterior and posterior end of this developmental stage. The eight papillae are arranged in two concentric circles consisting of two types of morphologically different receptors. Accessory nerve processes end free in the tip of the head. The paired amphids contain nine dendritic processes and accessory axons are seen in the surrounding cells. The basic structure of the amphids and of the circumoesophageal nerve ring is similar to that of other filarial nematodes. Two presumably neurosecretory cells are associated with the nerve ring. The reticular cytoplasm of these cells merges with the rough-surfaced endoplasmic reticulum of the lateral hypodermal chord. The paired phasmids at the posterior end of the developmental stages consist of single modified cilia that are embedded in an electron-dense mass. The receptor cell has access to the outside by a channel ending with a cuticular pore.

Morphological demonstration of essential functional changes after in vitro and in vivo transition of infective Onchocerca volvulus to the post-infective stage.

Transmission electron microscopic investigation of the morphogenesis of Onchocerca volvulus through the third moult to the post-infective stage revealed essential alterations in various larval organs. Complete rebuilding of surface structures, the reduction of secretory granules in the glandular oesophagus, the unfolding of the intestine, an increase in the number of nerve fibres in the nerve ring and novel sensory papillae were significant findings. Transition from third- to fourth-stage larvae (L4) started as early as 48 h after transfer to vertebrate conditions in vivo in surrogate hosts and in vitro. After a resting period of about 60 h to enable a reduction in gland size and the loosening of the old cuticle and formation of the new one, the larvae started to cast the infective-stage cuticle. Young L4 exhibited a thin, monolayered cuticle and did not rebuild a glandular oesophagus. The body cavity widened, the intestine unfolded and the increased number of microvilli indicated the resumption of metabolic activity.

Eosinophil-larval-interaction in onchocerciasis: heterogeneity of in vitro adherence of eosinophils to infective third and fourth stage larvae and microfilariae of Onchocerca volvulus.

Adherence of eosinophilic granulocytes from patients with onchocerciasis to microfilariae (Mf), third (L3) and fourth (L4) stage larvae of Onchocerca volvulus was studied in vitro. Native and heat-inactivated sera from patients with onchocerciasis (OS), from endemic controls without signs of the disease (ECS), from healthy Caucasians (NS) or foetal calf serum (FCS) served as sources for adherence mediating factors. In FCS-supplemented medium eosinophils did not adhere to any larvae. None of the sera mediated the adherence of eosinophils to L4. Eosinophils adhered to L3 in the presence of OS, ECS and NS, whereas OS exclusively mediated adherence to Mf. Reduced adherence rates of eosinophils to L3 occurred in heat-inactivated or zymosan-activated OS, ECS or CS. Eosinophils bound to the L3 cuticle of moulting stage but not to the newly exposed L4 cuticle. A single adherent layer of effector cells was found around cast L3 cuticle, multiple layers were found around intact L3 leading to subsequent paralysis of the larvae and to an amplification of the toxic effector potential by homotypic intereosinophilic adhesion. Our experiments document heterogeneity of in vitro effector cell adherence to the three larval stages of O. volvulus and indicate that complement-dependent as well as independent mechanisms are operative in eosinophil-larval-interaction. The results emphasize the importance of the invading infective larval stages of O. volvulus as possible targets for vaccine production.

Morphological alterations of male Onchocerca volvulus after in vitro exposure to mel w and milbemycin a confirming the results of viability tests.

The suitability of two viability parameters used for screening of antifilarial activities of new compounds was examined by parallel observation of the morphology. Male Onchocerca volvulus were exposed in vitro to 10 mumol mel w and milbemycin a and examined by light and transmission electron microscopy. The viability was assessed by measurement of the motility, using a micromotility meter and by determination of tetrazolium reduction. Already twelve hours after exposure to mel w the muscles of the body wall showed severe damage. After 36 hours the other tissues revealed degenerative changes and after 60 hours disintegration of all tissues was observed. Effects on the morphology caused by milbemycin a were seen earliest after 60 hours. Condensed cytoplasm in the hypodermal layer and beginning degeneration of spermatogenic stages indicated drug activity. The time-point of appearance of these drug induced morphological alterations was in accordance with the decrease of the motility indices and the degree of tetrazolium reduction. Morphological alterations indicating irreversible damage of worm tissues are a reliable parameter to detect macrofilaricidal activity. The good agreement between the results of the morphological examination and the assessment of the motility and the tetrazolium reduction confirms the suitability of the latter two assays for in vitro drug screening with O. volvulus.

Ultrastructural study of the interaction between eosinophilic granulocytes and third and fourth stage larvae of Onchocerca volvulus.

The interaction in vitro between eosinophil effector cells and third and fourth stage larvae of Onchocerca volvulus was studied by electron microscopy. The morphological observations demonstrated different mechanisms of attack of eosinophil cells that are dependent upon the time of incubation. Rapid adherence to the cuticle of the target, flattening, secretion of granule contents, vacuole formation and, finally, complete degranulation of the eosinophils were seen after incubation with third stage larvae and moulting stages. Alterations of the epicuticular and cuticular structures could be found near the attachment site of the cells. The eosinophils, however, showed no interactions with fourth stage larvae of this filarial parasite.

In vitro assessment of the activity of anthelmintic compounds on adults of Onchocerca volvulus.

The viability of adult Onchocerca volvulus and the effect of 12 known anthelmintic compounds on the parasites have been evaluated in an in vitro culture system. Three different parameters, a colorimetric assay, using NADH-dependent reduction of a tetrazolium salt to dark blue formazan by living adult worms, motility indices of male worms and lactate excretion of female worms were used to determine worm viability. The experiments showed that over a short term period of six days the viability of the worms did not decline significantly. The use of males isolated by dissection of whole nodules for the evaluation of drug effects in vitro is preferable to collagenase isolated worms. Mel W, milbemycin a and d, ivermectin, levamisole, CGP 6140 and, to a lesser extent, suramin immobilized male worms or significantly reduced the motility indices at a concentration of 10 microM. The tetrazolium reduction by male worms was not affected by levamisole, whereas the other active compounds demonstrated significant inhibitory effects. Diethylcarbamazine, mebendazole, flubendazole, metrifonate and CGP 20376 had no significant effect on male viability. Comparable activity was seen with the intact female worms isolated by collagenase digestion. Mel W, the milbemycins and ivermectin significantly inhibited tetrazolium reduction, whereas suramin and the other compounds had only slight or no inhibitory effects on female O. volvulus. Although one still has to aim at an improvement of the culture conditions, the in vitro test system using adult O. volvulus provides a basis for further research on potential antifilarial compounds.

A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50% loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic studies on the effects of CGP 6140 and CGP 20376 on microfilariae and third stage larvae of Onchocerca volvulus.

Electron microscopic examination was used to assess the effects of CGP 6140 (proposed generic name: amocarcine) and CGP 20376 (proposed generic name: metobethiamide) on the fine structure of microfilariae and third stage larvae of Onchocerca volvulus after in vitro exposure to different concentrations of these compounds. The range of concentrations was selected according to the expected plasma levels that may be reached with these drugs. The microfilariae showed distinct effects on the muscle cells as myelin figures, vacuolisation and disintegration of the cytoplasm after both compounds. The third stage larvae showed the same effects on the muscle cells including changes in the mitochondria and the hypodermal tissue. In addition, the glandular esophagus demonstrated degenerative alterations of the cytoplasm and loosening of the cuticular lining after exposure to CGP 6140. CGP 20376 led to severe damage of the fibres of the nerve ring. No degenerative changes of the microtubuli were observed. Comparison of the morphological effects observed by electron microscopy with the reduction rates of motility and moulting recorded in the in vitro tests showed good correlation between these different parameters. The observed effect on the fine structure was the most sensitive parameter.

Studies on the activity of the Ciba Geigy compounds CGP 6140, 20376, 20309 and 21833 against third and fourth stage larvae of Onchocerca volvulus.

The effect of four new antifilarial compounds, (CGP 6140, 20376, 20309 and 21833, Ciba Geigy Ltd. Basle) on the viability of third and fourth stage larvae of Onchocerca volvulus was tested in vitro and in vivo. The motility of the larval stages and the moulting process from the third to the fourth stage were used as parameters for the drug activity in vitro. All four compounds showed good effects in vitro on the moulting rates compared to the controls after incubation at different concentrations in culture medium with serum. After an exposure for three hours to different concentrations of the compounds in medium without serum, only CGP 20376 had a clear inhibitory effect on the developing larvae. The moulting rate was reduced by this compound to 20% of the control values with 1 microgram/ml, a concentration that is comparable to plasma levels reported from chimpanzees. In both experimental situations, with serum and without serum, the motility of the developing larvae was only affected at very high concentrations of the compounds. The results obtained with fourth stage larvae were comparable. CGP 6140 and CGP 20376 were tested in vivo, using a diffusion chamber technique for the implantation of infective larvae into the peritoneum of Mastomys natalensis and assessing the survival of the implanted parasites. High doses of CGP 6140 had only unsatisfactory effects on the larvae in this model system whereas CGP 20376 had inhibited moulting and killed most larvae with a daily dose of 25 mg/kg for five consecutive days.

In vitro study on the effect of the new chemotherapeutic agents CGP 6140 and CGP 20376 on the microfilariae of Onchocerca volvulus.

The effect of two new filaricidal compounds, CGP 6140 and CGP 20376, on the microfilariae of O. volvulus was tested in vitro. Culture medium consisted of a 1:1 mixture of IMDM/NCTC 135 supplemented with 20% heat-inactivated foetal calf serum and gentamycin. For pulse experiments the microfilariae were exposed to the drugs for one or three hours in medium without serum and then replaced to serum supplemented medium. In the continuous cultures the microfilariae were exposed to the drugs fur up to 72 hours without a change of medium. The effect of the drugs was assessed by their ability to reduce the larval motility. A reduction of motility of 98-100% after 24 hours of exposure occurred at levels of 1 micron/ml CGP 20376 or 10 mu mg/ml CGP 6140. After exposure for one hour CGP 20376 immobilized 100% of the microfilariae irreversibly at a drug level of 1 microgram/ml. Whereas no significant immobilization was seen with CGP 6140 after one hour. After exposure for three hours CGP 6140 immobilized up to 100% of the microfilariae at drug levels of 10 and 5 micrograms/ml.

Development of infective larvae of Onchocerca volvulus in diffusion chambers implanted into Mastomys natalensis.

Diffusion chambers containing vector-derived infective larvae of O. volvulus were implanted into male Mastomys natalensis and removed after periods up to 100 days. Nearly all chambers contained motile living parasites. After two weeks lengths and diameters of the larvae had increased significantly and after 100 days one juvenile worm showed well developed papillae at the posterior end.