Some hydrolase activities from the tick Hyalomma lusitanicum koch, 1844 (Ixodoidea: Ixodida).

In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

Cholinesterase and phosphatase activities in adults and infective-stage larvae of levamisole-resistant and levamisole-susceptible isolates of Haemonchus contortus.

Cholinesterase (ChE) and acid phosphatase (AP) activities, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of adult and infective-stage larvae of levamisole-resistant and levamisole-susceptible Haemonchus contortus. In contrast to other gastrointestinal nematodes, the ChE activity was higher in L3 than in adults and, in both cases, was mainly associated with membranes. ChE activity was inhibited by Triton X-100 and was only detected in membrane-bound fractions when the detergent was removed. Differences between resistant and susceptible L3 were observed in the response to inhibitors (cytosolic fraction) and in the enzymatic content (membrane-bound fraction). Phosphatase activity was detected at acidic pH in all fractions, being higher in the adult than in the L3 stage. In the former, most of the enzyme was localized in the membrane-bound fractions, whereas in the latter it was mainly in cytosolic fractions. This difference could be correlated with the activity in the gut. In inhibition assays, a difference between cytosolic fractions from resistant and susceptible adults was observed in their response to 1 mmol/L tartaric acid.

Trichomonas vaginalis: determination of acid phosphatase activity as a pharmacological screening procedure.

A simple method to screen trichomonacides, based on the quantification of acid phosphatase (AP) activity, has been designed. Using p-nitrophenyl phosphate as chromogenic substrate, we first determined the optimal conditions for enzyme reaction. After seeding, a linear correlation between number of trichomonads and optical densities at 405 nm was obtained at 24 hr but not at 48 hr. Then, the inhibitory effect of metronidazole was assessed both by microscope counts and by AP determination. Similar values for 50% inhibitory concentrations (2.6 microM), with 95% confidence limits of 1.91-3.33 for microscopic and 2.21-3.05 for colorimetric method, were obtained. We concluded that the colorimetric method described in this investigation is suitable for pharmacological studies and for the screening of new, potential antitrichomonal agents.

Nematocidal activity of nitazoxanide in laboratory models.

The nematocidal activity of a broad-spectrum antiparasitic agent, nitazoxanide [( N-(5-nitrothiazol-2-gammal)salicylamide; NTZ], was evaluated in both in vitro and in vivo models using Caenorhabditis elegans, Heligmosomoides polygyrus and Trichinella spiralis. In vitro, NTZ (100 muM) exhibited a low activity against C. elegans and had no effect on embryonation and hatching of H. polygyrus eggs. At concentrations of 100 and 50 muM, the inhibition of excretion/secretion of acetylcholinesterase and acid phosphatase of adult H. polygyrus by NTZ was variable. The in vitro effects of mebendazole (5 muM), albendazole (1 muM) and levamisole (10 muM) were superior to those of NTZ. In mice, NTZ at 1 g/kg proved to be inactive against preadults of T. spiralis whereas mebendazole at 10 mg/kg reduced the worm burden by up to 83%. NTZ at 1 g/kg per day for 3 consecutive days showed a low activity against adults of H. polygyrus (21% reduction). Levamisole, at a single dose of 10 mg/kg, reduced the worm burden by up to 89.9%. The results of this study suggest that NTZ would not have met criteria of a candidate compound.

Acid phosphatase activity in excretion/secretion products from Heligmosomoides polygyrus adults: an indicator of the physiological status of the worms.

Acid phosphatase (AP) activity was detected in 24 h culture media from adult Heligmosomoides polygyrus. Female and male excretion/secretion products showed similar specific activity. For both, the AP had a pH optimum of 4.0 and was inhibited by sodium fluoride, tartaric acid, and sodium orthovanadate. The release of AP by adult worms was significantly inhibited by adverse incubation conditions (temperatures of 20 degrees C and 4 degrees C), known physiological perturbers ( t-butylhydroperoxide and sodium azide), and broad spectrum anthelmintics (albendazole, levamisole, morantel, and ivermectin). These results indicate that the AP activity level in the culture medium may be an indicator of the physiological status of the worms.

The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics.

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated. The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.

Nematocide activity of 6,7-diarylpteridines in three experimental models.

The in vitro nematocide activity of seventeen 6,7-diarylpteridines has been tested using three different experimental models, Caenorhabditis elegans, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. The method of evaluation of inhibition in the secretion of acetylcholinesterase by H. polygyrus seems to be the most indicated to avoid false positives. The in vivo activities, against Trichinella spiralis, of the most in vitro active pteridines have been assayed. All pteridine derivatives bearing 6,7-di-p-bromophenyl substituents have shown in vitro nematocide activities in the three experimental models used. Amongst all the pteridines tested in vivo, only 2,4-pteridinedithione derivatives exhibited moderate activity.

[Comparative activity of several antibiotics against Toxoplasma gondii in a mouse model]. / Actividad comparada de varios antibióticos frente a Toxoplasma gondii en un modelo murino.

BACKGROUND: Infections with Toxoplasma gondii poses a great hazard to immunocompromised patients and to pregnant women. Currently, the recommended therapy for toxoplasmosis is the synergistic combination of pyrimethamine and sulfadiazine (P/S). However, this therapeutic regimen may be toxic or only partially effective. Because pyrimethamine is potentially teratogenic, its use during the early months of pregnancy is not recommended. There is a critical need for newer and safer compounds for the treatment of toxoplasmosis. These considerations led us to evaluate other potential therapeutic agents--minocycline, clindamycin, roxithromycin, azithromycin, clarithromycin and miocamycin--as well as comparison of P/S and spiramycin efficacy against T. gondii. METHODS: The activity of antibiotics was tested in a murine model of acute toxoplasmosis. NMRI mice were infected intraperitoneally with 200 tachyzoites of the highly virulent RH strain of T. gondii, and treated, every 12 h or 24 h, for ten consecutive days. RESULTS: P/S (a dose of 4/250 mg/kg of body weight per day), minocycline (200 mg/kg) and azithromycin (200 mg/kg) were the most active compounds (100% of mice survival). Clindamycin, roxithromycin, spiramycin and clarithromycin were less effective. Miocamycin was not effective in this model. CONCLUSIONS: Our findings support the view that minocycline and azithromycin could be useful in many cases of toxoplasmosis. Clinical studies are needed to determinate the relative efficacy and safety of these antibiotics for the treatment of toxoplasmosis in humans.

Styryl and azastyryl 1,3-benzazoles with antihelminthic activity.

New 2-benzylideneimino- and 2-styryl-1,3-benzimidazole and benzothiazole derivatives have been prepared and tested in vitro against Caenorhabditis elegans, and Heligmosomoides polygyrus, showing some of them, interesting properties as inhibitors, which were not observed in the complementary in vivo tests. In order to rationalize the activity, log P was measured for all compounds, and QSAR models were developed, allowing the optimisation of the in vitro activity.