Comparative effects of anthelmintics on c-MDH fromMolinema dessetae (Nematoda: Filarioidea) and from a mammal

Malate dehydrogenase (MDH) (E.C.1.1.1.37) activity was detected in the filariaMolinema dessetae at a level similar to those found in other filariae. InM. dessetae, the cytoplasmic form (c-MDH) predominated and the study was performed on partially purified fractions. The pH optimum for oxaloacetate reduction was 6.1, with maximal activity at 7811 nmol min^–1 mg protein^–1, but high concentrations of oxaloacetate inhibited MDH activity. TheK _m value for oxaloacetate was determined as 22 M forM. dessetae c-MDH and 33M for mammalian c-MDH. Anthelmintic drugs were compared as potential inhibitors of filarial and mammalian c-MDH. Among the compounds evaluated, amocarzine showed a specific inhibitory effect on filarial c-MDH through only at high concentrations. Suramin alone showed an inhibitory effect at low concentrations (K _i=1.15 M) but without selective action towards filarial c-MDH. The suramin type of inhibition was found to be competitive. Suramin probably acts on both enzymes in the same manner. Nevertheless,M. dessetae c-MDH is proposed as a suitable enzyme assay model to screen MDH inhibitors as potential filaricides.     

Cytochemical characterization of the third-stage larva of Wuchereria bancrofti (Nematoda: Filarioidea)

In the present paper, we report the results we obtained using several cytochemical techniques to analyze the infective larva of Wuchereria bancrofti. An imidazole osmium tetroxide solution was used to visualize unsaturated fatty acids. A highly contrasted material forming a continuous structure was observed on the larval surface and over the epicuticle. A strong reaction was observed on the esophagus and also on the inner secreted material. Carbohydrates containing vic-glycol groups were not observed on the cuticle of the third-stage larva of W. bancrofti submitted to the Thiéry technique. Using a panel of eight gold-labeled lectins, we found that the cuticle exhibited slight labeling with all lectins used, indicating residues of N-acetyl-d-glucosamine, N-acetyl-galactosamine, d-galactose, d-manose, and l-fucose. Surface anionic sites were visualized by using cationized ferritin particles. Treatment with trypsin partially inhibited the reaction, whereas the treatment with chondroitinase ABC, a specific enzyme for glycosaminoglycans, completely abolished the labeling with cationic particles.     

Histology of adultLitomosoides carinii (Nematoda: Filarioidea)

The histology of male and femaleLitomosoides carinii was studied in serial sections. The body wall of the female worm consists of a rather thin cuticle, very extended lateral hypodermal chords and small sectors of shallow coelomyarian muscle cells. In the female worm an intestinal lumen is found, which is filled with material throughout the worm's length. Both ovaries are situated close to the posterior end. In both genital tubes all stages of oogenesis and embryogenesis could be observed in approximately equal stages of development in most of the sections. The lateral chords of the male worm are very small, whereas the somatic muscle cells are large and of the circomyarian type. The intestine is a minute tube beside the much thicker testis and vas deferens. Considerable sexual dimorphism could be observed in almost all organs of adultLitomosoides carinii.     

In vitro-Versuche mitLitomosoides carinii (Nematoda: Filarioidea)

Zusammenfassung Setzt man in vitro fötales Kälber-Serum oder Human-Serum zum Grundmedium Tc 199 zu, so geben adulte Weibchen vonLitomosoides carinii gleich viele Mikrofilarien ab wie bei Zusatz von Baumwollratten-Serum. In Medien mitMeriones- bzw.Mastomys-Serum werden dagegen nur wenige Mikrofilarien freigesetzt und beiMastomys-Serum zudem noch immobilisiert. Hält man in vitro männliche und weibliche Würmer gemeinsam, so werden ebensoviele Mikrofilarien wie bei Einzelhaltung von Weibchen freigesetzt. Vier Wochen alte (d.h. ca. 2 Wochen vor der Kopulation befindliche) Wurmpaare kopulieren in vitro in Tc 199 mit 33% Baumwollratten-Serum nicht (keine Insemination der Weibchen). Die Weibchen überleben jedoch 18 Tage (harmonisches Mittel) und wachsen dabei durchschnittlich um 5 mm (28%) in die Länge, die Männchen rollen das Hinterende in charakteristischer Weise ein. Werden 5 oder 6 Wochen alte Würmer für 4 bis 6 Tage paarweise in vitro gehalten und danach zu mehreren Paaren in Baumwollratten implantiert, so wachsen sie in vivo auf normale Länge heran, kopulieren und schütten Mikrofilarien ins zirkulierende Blut aus. Weibchen, die zur Kontrolle allein implantiert werden, wachsen ebenfalls heran, bleiben jedoch steril, d.h. in vitro findet keine Insemination statt.

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Experiments in vitro withLitomosoides carinii (Nematoda: Filarioidea)II. Influence of different sera on the release of microfilariae and experiments on the readiness for copulation of the worms

When fetal calf serum or human serum is added to the culture medium Tc 199, adult females ofLitomosoides carinii release the same amount of microfilariae in vitro as after addition of cotton rat serum. In contrast, in media with serum ofMeriones unguiculatus orMastomys natalensis only a few microfilariae are released; moreover, addition ofMastomys serum immobilizes them. When males and females are kept together, the same amount of microfilariae is released as compared with single females. Worms 4 weeks of age (i.e., about 2 weeks before copulation) maintained in medium Tc 199 with 33% cotton rat serum do not mate (no insemination of the females). However, the females live 18 days (harmonic mean) in vitro and grow on average 5 mm in length (28%) and the males roll up their tails in characteristic manner. Worms 5 or 6 weeks of age kept in pairs for 45 to 6 days in vitro were implanted into cotton rats. In vivo they grew to normal length, copulated and delivered microfilariae into the circulating blood. Females implanted solely into the recipient grew as well but remained sterile; this means that no insemination took place in vitro.

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Teil einer Dissertation an der Fakultät Biologie der Universität Tübingen. Mit teilweiser Unterstützung der Filariasis Component der UNDP/Weltbank/WHO Special Programme for Research and Training in Tropical Diseases     

Studies on Setaria cervi (Nematoda: Filarioidea)

Summary Living worms of Setaria cervi were successfully transplanted into the peritoneal cavity of white rats. The worms produced microfilariae which were recovered from the peripheral circulation in fairly good numbers upto a maximum period of six to seven weeks. The microfilariae showed a definite periodicity with two high peaks in every 24 hours, one between 6 to 8 A.M. and the other between 6 to 8 P.M. The two lowest levels were obtained at mid-day and mid-night respectively. The maximum density of the microfilariae was, on the average, seen somewhere between the 3rd. and the 5th. week after infection. The longest period a transplanted worm was demonstrated to survive was 45 dyas.With 2 Figures in the Text     

Fine structure of adultLitomosoides carinii (Nematoda: Filarioidea)

The fine structure of the body wall and the intestine of male and femaleLitomosoides carinii was studied in sections through the middle and posterior regions of the worms. In the sublateral cells of the female hypodermis the organelles are arranged in regular layers. The zone with the basal labyrinth is the most prominent layer. The intestinal epithelium of the female worm varies considerably in thickness. The cytoplasm of these cells contains many large droplets. The lateral hypodermal chords of the male worm are very narrow bands. The muscle cells of the male worm are of the circomyarian type, without an afibrillar portion. The intestinal lumen of the male worm is very narrow, and the epithelial cells contain very large droplets. In both sexes the hypodermis contains bacteria. Larger glycogen deposits were absent in the hypodermal chords and in the muscle cells of the midbody regions.     

Evaluation of a protocol for lactate dehydrogenase (LD) isoenzymes

Two divergent laboratory approaches to the measurement of lactate dehydrogenase (LD) isoenzymes (LDIs) were evaluated. Abnormal values for total creatine kinase (CK), CK-MB, total LD, and LDIs were greater than 250 U/L, greater than or equal to 5% total CK, greater than 180 U/L, and LD1/LD2 ratio greater than 1.0, respectively. The first approach, measuring LDI on all requests regardless of total LD activity, showed the following results: 161 LDI specimens, 63 CK-MB + (LD1 greater than LD2, 6; LD1/LD2 normal, 57); 98 CK-MB - (LD1 greater than LD2, 5, LD1/LD2 normal, 93). Medical chart review of the 98 CK-MB negative specimens, representing 44 patients, showed that in seven patients with acute myocardial infarction (MI), none would have been missed clinically even if LDIs had not been done, and in only one case did LDIs play a confirmatory role in a patient who persistently had CK-MB-negative results but who had a strong clinical suspicion of MI. None of the 42 patient specimens with normal total LD activity had an LD1 greater than LD2. The second approach, measuring LDIs only if requested, only if total CK was abnormal, only if results for CK-MB were negative, and only if total LD was abnormal, showed the following results: 71 LDI specimens were not done (46 with normal total LD, 25 CK-MB + specimens). Medical chart review of the 71 LDIs not determined, representing 38 patients, showed that none of the 14 patients with the final diagnosis of MI would have been clinically missed, even if LDI had not been done, and in only one instance did LDIs confirm the clinical suspicion of MI in a patient with previously negative results for CK-MB. The LDI protocol represented by the second approach has been shown to be an effective means of identifying those occasional patients who require this confirmatory test, in whom the CK-MB results are negative but in whom there is a strong clinical suspicion of MI.     

In vitro-Versuche mit Litomosoides carinii (Nematoda: Filarioidea)

Zusammenfassung Bei täglichem Wechsel des Kulturmediums (Tc 199) und Luft als Gasphase entwickeln sich die Embryonen von Litomosoides carinii intrauterin zu Mikrofilarien weiter und werden innerhalb vom 5–6 Tagen vollständig in das Medium freigesetzt. Die Oogenese bzw. eine weitere Befruchtung von Eiern findet jedoch in vitro bei allen von uns untersuchten Medien nicht statt. Im Mittel stößt ein Weibchen in vitro während 5–6 Tagen 140 × 10³ Mikrofilarien/Tag aus. Initial beobachtet man pro Tag durchschnittlich 300 × 10³ Mf/.Zusatz von Pferde-Serum hemmt die Freisetzung von Mikrofilarien in vitro, Baumwollratten-Normalserum verlängert die Überlebenszeit der weiblichen Würmer, erhöht jedoch nicht die Gesamtzahl freigesetzter Mikrofilarien bzw. zurückgehaltener Embryonalstadien. Serum von postpatenten Tieren hat keinen Einfluß auf die Zahl freigesetzter Mikrofilarien oder auf deren Vitalität und die Überlebenszeit der Weibchen.In vitro freigesetzte Mikrofilarien leben in Tc 199+33% Baumwollratten-Normalserum und Luft als Gasphase bei täglichem Wechsel des Mediums länger als 8 Tage. Aus dem Blut patenter Tiere isolierte Mikrofilarien leben jedoch höchstens 6 Tage, bei 48stündigem Wechsel des Mediums nur 4 Tage.

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Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea)I. Maintenance of adult females and microfilariae as well as release of microfilariae in different culture media

Summary Embryos of L. carinii continue intrauterine development to microfilariae and are totally released into the medium within 5–6 days when the latter (Tc 199) is changed daily and air is used as the gas phase. Oogenesis or further fertilization of eggs, however, does not occur in vitro in any of the media examined by us. One female releases 140 × 10³ microfilaria/day on an average in vitro within 5–6 days. Mean initial numbers of 300 × 10³ Mf//day are observed.Addition of equine serum inhibits microfilarial release in vitro; normal cotton rat serum prolongs survival of females while total numbers of released microfilariae or retained embryonic stages are not increased. The serum of post-patent animals does not influence the numbers of released microfilariae or their viability or survival of females.Microfilariae released in vitro in Tc 199+33% normal cotton rat serum survive for more than 8 days, when air is used as the gas phase and the medium is changed daily. Microfilariae isolated from the blood of patent animals survive for at most 6 days, at a 48-hourly change of medium survival does not even exceed 4 days.

     

Cellular H- and M-type lactate dehydrogenase (LDH) isoenzymes and tumour diagnosis--an immunohistochemical assessment.

Cellular H- and M-type lactate dehydrogenase (LDH) isoenzymes were determined immunohistochemically in a wide range of normal, inflammatory, benign neoplastic, and malignant tissues to assess the possible value of the isoenzymes in tumour diagnosis. Both types of LDH were detected almost ubiquitously in normal tissues. Increased staining intensity of LDH, especially M-type LDH, was demonstrated in most types of malignant tumours studied. Similar increased staining intensity of the isoenzymes was also noted to parallel the proliferative activity of the cells in non-neoplastic diseases and benign neoplasms. The results suggest that changes of cellular H- and M-type LDH isoenzymes are not specific for any type of malignant tumours. The increase in cellular H- and M-type LDH isoenzymes appears to be related to proliferation of cells in general.     

Effects of albendazole on Litomosoides chagasfilhoi (Nematoda: Filarioidea) females in vivo

Gerbils (Meriones unguiculatus) experimentally infected with Litomosoides chagasfilhoi were treated with a single oral dose of 40 or 80 mg of albendazole, respectively. Observation of the microfilaremia after the treatment showed that both single oral doses of albendazole decreased the microfilaremia in L. chagasfilhoi infection. The body wall was composed of a cuticle, a hypodermis, and a muscular layer, and treated nematodes showed no morphological alterations. The ultrastructural alterations produced by treatment with 40 mg of albendazole included a higher number of membrane invaginations in the basal labyrinth of the uterine epithelium and the presence of myelin figures in this region. Inside the uterus, most embryos and microfilariae were disintegrated. The treatment with 80 mg of albendazole did not produce alterations in the uterine wall, and the number of vesicles near the microfilariae sheath was smaller than that observed in the untreated and in the 40-mg treatment groups. However, all the microfilariae observed in the uterus were extensively damaged with cytoplasmic vacuolization and cellular degeneration. No alterations in the intestinal cells were observed after treatment with 40 or 80 mg of albendazole. The present study contributes to the knowledge of albendazole’s effects in filariids and demonstrates the potential embryotoxic and microfilaricidal consequences of this drug.     

Expression and characterization of lactate dehydrogenase from Schistosoma japonicum

Some effective antiparasitic drugs for chemotherapy schistosomiasis, such as praziquantel and artemether, are proved to act on lactate dehydrogenase (LDH) in vitro, but detailed molecular action mechanisms of these have not yet been elucidated. To this end, the sequence encoding LDH of Schistosoma japonicum (SjLDH) was amplified from the cDNA library of the adult parasite. Sequence analysis revealed an open reading frame (ORF) of 999 bp, encoding 332 amino acids with a molecular weight of 36.120 kDa. It has three transmembrane regions and may be located in the cytoplasm membrane. The encoding ORF was inserted into pET-28a vector and expressed in Escherichia coli (E. coli) induced by IPTG. A 36-kDa protein with 6×His-tag was purified by metal affinity column and identified by SDS-PAGE and Western blot, which had high LDH activity of 379 U/mg. Characterization such as Michaelis constant (K _m) and maximum velocity (V _max), optimum pH, and temperature of the protein were assayed. It provides a model for drug screening on SjLDH.     

Electron microscope observations on Onchocerca ochengi and O. fasciata (Nematoda: Filarioidea)

The fine structure of the females and males of Onchocerca ochengi (parasitizing zebu and cattle) and of the females of O. fasciata from camels were described and compared to other filariae of the genus Onchocerca. It was shown that O. ochengi resembles O. volvulus of humans in its degree of development, while being more primitive than O. gibsoni. Besides other similarities O. ochengi attracts inflammatory cells in the way of O. volvulus and these could be a model for chemotherapeutic trials. Received: 15 January 1997 / Accepted: 1 February 1997     

Scanning electron microscopy (SEM) of some Setaria species (Filarioidea,Nematoda)

Summary Some Setaria spp., such as S. digitata, S. marshalli, S. marshalli pandei, S. equina and S. labiatopapillosa were studied by scanning electron microscopy at the critical points in the anterior and posterior parts of the adult worms. The amphids. Deirids, fine transverse striations of the cuticle, and phasmidial pore, as well as the transverse bands (lugae), postdeirid and ventral papillae (only of the male) were visualized clearly at this examination. They contribute towards the identification of Setaria spp., in addition to the characteristics which are demonstrated by common light microscopy.The main part of this paper was presented to the Third International Congress of Parasitology, held in Munich, Sect. B 5(20), 1974.Guest researcher.     

Fecundity ofLitomosoides carinii (Nematoda,Filarioidea) in vivo and in vitro

Several parameters concerning the reproduction ofLitomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 18±2 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7–8 weeks p.i. the females were 71±6 mm long and on average contained 308×10³ embryos/female, of which 19% were pathologically altered. In the middle of patency 16–20 weeks p.i. the females had grown up to 100±11 mm in length and now contained 509×10³ embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a femaleL. carinii in vivo of around 20×10³ microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.Dedicated to Dr. B.O.L. Duke on the occasion of his 60th birthday