Imidazole-containing phthalazine derivatives inhibit Fe-SOD performance in Leishmania species and are active in vitro against visceral and mucosal leishmaniasis.

The in vitro leishmanicidal activity of a series of imidazole-containing phthalazine derivatives 1-4 was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, and their cytotoxicity on J774·2 macrophage cells was also measured. All compounds tested showed selectivity indexes higher than that of the reference drug glucantime for the three Leishmania species, and the less bulky monoalkylamino substituted derivatives 2 and 4 were clearly more effective than their bisalkylamino substituted counterparts 1 and 3. Both infection rate measures and ultrastructural alterations studies confirmed that 2 and 4 were highly leishmanicidal and induced extensive parasite cell damage. Modifications to the excretion products of parasites treated with 2 and 4 were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds 2 and 4 were potent inhibitors of iron superoxide dismutase enzyme (Fe-SOD) in the three species considered, whereas their impact on human CuZn-SOD was low. Molecular modelling suggests that 2 and 4 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the antioxidant features of the enzyme.

Prospects of an alternative treatment against Trypanosoma cruzi based on abietic acid derivatives show promising results in Balb/c mouse model.

Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is an example of extended parasitaemia with unmet medical needs. Current treatments based on old-featured benznidazole (Bz) and nifurtimox are expensive and do not fulfil the criteria of effectiveness, and a lack of toxicity devoid to modern drugs. In this work, a group of abietic acid derivatives that are chemically stable and well characterised were introduced as candidates for the treatment of Chagas disease. In vitro and in vivo assays were performed in order to test the effectiveness of these compounds. Finally, those which showed the best activity underwent additional studies in order to elucidate the possible mechanism of action. In vitro results indicated that some compounds have low toxicity (i.e. >150 µM, against Vero cell) combined with high efficacy (i.e. <20 µM) against some forms of T. cruzi. Further in vivo studies on mice models confirmed the expectations of improvements in infected mice. In vivo tests on the acute phase gave parasitaemia inhibition values higher those of Bz, and a remarkable decrease in the reactivation of parasitaemia was found in the chronic phase after immunosuppression of the mice treated with one of the compounds. The morphological alterations found in treated parasites with our derivatives confirmed extensive damage; energetic metabolism disturbances were also registered by (1)H NMR. The demonstrated in vivo activity and low toxicity, together with the use of affordable starting products and the lack of synthetic complexity, put these abietic acid derivatives in a remarkable position toward the development of an anti-Chagasic agent.

In vitro leishmanicidal activity of pyrazole-containing polyamine macrocycles which inhibit the Fe-SOD enzyme of Leishmania infantum and Leishmania braziliensis species.

The in vitro leishmanicidal activity and cytotoxicity of pyrazole-containing macrocyclic polyamines 1-4 was assayed on Leishmania infantum and Leishmania braziliensis species. Compounds 1-4 were more active and less toxic than glucantime and both infection rates and ultrastructural alterations confirmed that 1 and 2 were highly leishmanicidal and induced extensive parasite cell damage. Modifications in the excretion products of parasites treated with 1-3 were also consistent with substantial cytoplasm alterations. Compound 2 was highlighted as a potent inhibitor of Fe-SOD in both species, whereas its effect on human CuZn-SOD was poor. Molecular modelling suggested that 2 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the enzyme`s antioxidant features.

Clinical use of oral antihistamines and intranasal corticosteroids in patients with allergic rhinitis.

BACKGROUND: Second-generation oral antihistamines (AH) and intranasal corticosteroids (ICS) are the most widely used drugs for allergic rhinitis (AR). OBJECTIVE: To obtain information on the preferences for and applications of these drugs under conditions of routine clinical practice. METHODS: We performed a multicenter multidisciplinary observational study. Participating physicians completed a questionnaire with information on preferences for and application of drugs for AR, patient characteristics, and physician/patient satisfaction with the treatment provided (visual analog scale). RESULTS: A total of 1008 physicians participated in the study (primary care physicians, 53%; ear, nose, and throat specialists, 28%; allergologists, 19%). Treatment preferences in AR were AH combined with ICS (7.68), AH (7.25), and ICS (6.94). AH and ICS were used continuously by 58% and 71% of patients, respectively. Physicians reported having a good knowledge of the Allergic Rhinitis and its Impact on Asthma guidelines (93%), and 90% claimed to follow the guidelines. A total of 4040 patients were recruited (52% females, mean [SD] age 34 [14] years). The findings for AR were as follows: mean (SD) duration, 9 (8) years; persistent AR, 52%; mild AR, 72%; moderate AR, 7%; and severe AR, 1%. Patients considered the disorder to be well controlled/almost controlled (79%). As for treatment, 77% followed the regimen recommended by the physician. Oral treatment (41%) and intranasal treatment (22%) were preferred, while 35% showed no preference for any given administration route. The treatments prescribed were AH combined with ICS (66%), AH (20%), ICS (11%), other antihistamines (4%), and other drugs (6%). Combination treatment was the preferred therapy, regardless of the type of rhinitis. CONCLUSIONS: Physicians prefer and more often use combination treatment with oral AH and ICS, regardless of the frequency and intensity of AR.

Intestinal and haematic parasitism in the birds of the Almuñecar (Granada, Spain) ornithological garden.

Birds from the Almuñecar ornithological garden (Granada, Spain) were surveyed from June 2006 to May 2007 to establish programmes to prevent, control, and treat intestinal and haematic parasites. A total of 984 faecal samples and 41 samples of blood were collected from Psittacidae, Cacatuidae, Phasianidae, and Anatidae. One or more intestinal parasites were identified in 51.6% of the samples. Blood parasites were found in 26.8% of the birds examined. The most frequent pathogenic endoparasites were coccidians, such as Cyclospora sp. (4.5%), Eimeria sp. (4.1%) and Isospora sp. (2%) and helminths such as Capillaria sp. (10. 1%), Ascaridia sp. (4.9%) and Heterakis gallinarum (4.9%). All the parasites varied with season but the most were found year round. Multiple parasitic infections by intestinal parasites were common, with 196 of 984 faecal samples having 2-5 intestinal parasites. The most frequent cases of multiple parasitism were Blastocystis plus Entamoeba sp. and Blastocystis plus Cyclospora sp. The haematic protozoa detected were Haemoproteus sp. (17%) and Plasmodium sp. (7.3%). Multiple parasitism by Haemoproteus sp. and Plasmodium sp. was detected in 1 sample of Gallus gallus. After each sampling, some of the affected animals were treated according to our results, and the corresponding programmes of prevention and control were designed.

Intestinal parasitism in the animals of the zoological garden "Peña Escrita" (Almuñecar, Spain).

Gastrointestinal parasites cause serious diarrhoea in captive animals. Therefore, we have undertaken this study to establish programmes to prevent, control, and treat intestinal parasitism in the animals of the zoological garden "Peña Escrita" of Almuñecar (Granada). An annual survey was conduced to estimate the occurrence of gastrointestinal parasites and the seasonality of this parasitism. Between June 2006 and May 2007, 432 samples were collected from primates, carnivores, perissoodactyla, artiodactyla, rodentia, diprotodontia, galliformes, anseriformes and struthioniformes. One or more intestinal parasites were identified in 72.5% of the animals. The most frequent pathogenic endoparasites were Eimeria spp. (17.3%), Trichuris spp. (5.1%), Strongyloides spp. (4.5%), Cyclospora spp. (4.5%), Cryptosporidium spp. (3.2%) and Isospora spp. (2.6%). Iodamoeba butschlii, Parascaris equorum and Trichuris spp. did not vary with season and Cryptosporidium spp., Dicrocoelium dendriticum, Metastrongylus spp. and Cylicospirura spp. appeared exclusively in Artiodactyla. Multiple parasitic infections were common, 70% of animals presented with at least two parasites (maximum=6). The most frequent cases of multiple parasitism were Eimeria spp. plus Blastocystis spp. and Eimeria spp. plus Nematodirus spp., in the last case the animals presented explosive diarrhoea. In accord with our results, after each sampling, some of the affected animals were treated and the corresponding programmes of prevention and control were designed.

Prevalence of enteroparasites and genotyping of Giardia lamblia in Peruvian children.

Enteroparasites in children from three marginal urban districts of Trujillo (Peru) were studied to treat these children and to design a prevention and control programme. A total of 845 children were examined. The general prevalence of enteroparasites was of 66.3%, and 45.6% were multiparasitized. The pathogenic enteroparasite prevalence were 23.8% (Giardia lamblia), 4.6% (Iodamoeba buschlii), 2.6% (Cyclospora cayetanensis), 2.2% (Hymenolepis nana), and 2% (Cryptosporidium spp.). G. lamblia was the most frequent parasite both in diarrheic children (28.1%) as well as in nondiarrheic ones (19.5%). The G. lamblia genotypes were molecularly characterized by sequence analysis of the glutamate dehydrogenase (gdh) gene using PCR and RFLP. Sequence analysis revealed both Assemblage A (AI and AII) and Assemblage B (BIV), with the predominance of Assemblage AI. All the samples with Assemblage A were diarrheic but not those with Assemblage B. This is the first study of molecular characterization of G. lamblia in Peruvian children and confirms the importance of asymptomatic patients in the transmission of the giardiosis, especially in places with poor hygiene and sanitation.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases.

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment length polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10 percent IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71 percent at 48 h vs 14.5 percent), even after two weeks (47 percent vs 1.9 percent). Also, the percentage of extracellular stages augmented (25.3 percent vs 1.1 percent at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

Extracellular like-gregarine stages of Cryptosporidium parvum.

The present study confirms the existence of extracellular stages of Cryptosporidiumparvum during in vitro culture on MDCK, HCT 8 and Vero cells as well as alveolar macrophages, by optic, Nomarski and transmission electron microscopy images. Extracellular trophozoite/gamont, stages in syzygy, zygotes and spores with eight sporozoites were seen in the supernatant of the cultures. The first ultrastructural images of extracellular stages of C. parvum are shown in this study. The morphology of these stages, which have characteristics similar to those of some gregarines, support the contention that Cryptosporidium has closer affinity with gregarines. It also supports the necessity of reconsidering the life cycle of Cryptosporidium and the classification within the coccidia.

Purification and characterization of two iron superoxide dismutases of Phytomonas sp. isolated from Euphorbia characias (plant trypanosomatids).

Two superoxide dismutases (SODI and SODII) have been purified by differential centrifugation, fractionation with ammonium sulphate followed by chromatographic separation (ionic exchange and affinity), from a plant trypanosomatid isolated from Euphorbia characias, and then characterized for several biochemical properties. Both enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing superoxide dismutase. SODI had a molecular mass of approximately 66 kDa, whereas the molecular mass of SODII was approximately 22 kDa, both enzymes showing single bands. The isoelectric points of SODI and SODII were 6.8 and 3.6, respectively. The enzymatic stability persisted at least for 6 months when the sample was lyophilized and preserved at -80 degrees C. Digitonin titration and subcellular fractionation showed that both enzymes were in the cytoplasmic fraction, although part of SODII isoenzyme was also associated with glycosomes. We assayed these activities (SOD) in 18 trypanosomatid isolates on isoelectric focusing gels, and have demonstrated that the SOD is a biochemical marker sufficient to identify a trypanosomatid isolated from a plant as belonging to the genus Phytomonas and to distinguish between a true Phytomonas and other trypanosomatids that are capable of causing transient infections in plants.

In vitro culture of Glugea sp.

Recently the low host specificity of some microsporidians has been demonstrated and it has been indicated that many of these micro-organisms could be transmitted from invertebrates to mammals and adapt to changes in temperature. In this work, we demonstrate the first successful in vitro culture of a fish microsporidia of the genus Glugea on larval cells of the mosquito Aedes albopictus at 28 degrees C, and we show ultrastructural aspects of the different life cycle stages. It was impossible on salmon cells CHSE-214 at 21 degrees C. This study will be valuable for further work in biochemistry and immunology in addition to chemotherapy for microsporidiosis humans and animals.

Diet and organochlorine contaminants in women of reproductive age under 40 years old.

The diet of the breast-feeding mother impacts on the quality and quantity of the milk that she feeds her child. Milk can be a vehicle for toxins, such as drugs and their metabolites, viruses, nicotine, caffeine, alcohol, and organochlorine molecules such as PCBs, DDT, HCB, HCH and dioxins, which can harm the health of the breast-feeding child. The 24-h recall diet was considered appropriate to adequately study the diet of breast-feeding mothers and was used in the present preliminary study to establish the possible relationship between the food items consumed and the presence of pesticides in her milk. Two groups of randomly selected healthy breast-feeding volunteers aged between 17 and 35 years from two different areas were recruited: 34 from intensive agriculture zone, El Ejido (Almeria), from the "Hospital de Poniente" and 21 urban zone, the city of Granada, from the "Clinico" University Hospital. Application of the Spearman Correlation Test to the results from Almeria showed a certain positive correlation between the total intake of fats and both the p,p'DDD (rho=0.53, p< or =0.05) and methoxychlor (rho=0.48, p< or =0.05) in mature milk, and between the energy supplied by vegetables and the endosulfan-lactone in mature milk (rho=0.50, p< or =0.05). Among the group of breast-feeding women from Granada, there was a strong correlation between the intake of fats and both the p,p'DDT in transition milk (rho=0.90, p< or =0.05) and the p,p'DDD in mature milk (rho=0.90, p< or =0.05). In conclusion, there is a statistically significant relationship between the consumption of fatty foods and some organochlorine molecules and between the consumption of vegetables and pesticides, and the latter relationship occurs in Almeria but not in Granada.

In vitro evaluation of newly synthesised [1,2,4]triazolo[1,5a]pyrimidine derivatives against Trypanosoma cruzi, Leishmania donovani and Phytomonas staheli.

The antiprotozoal activity of newly synthesised compounds, all [1,2,4]triazolo [1,5a]pyrimidine derivatives, was tested against the protozoan parasites Trypanosoma cruzi, Leishmania donovani and Phytotmonas staheli. Six of these compounds significantly inhibited in vitro cell growth of the epimastigote forms of T. cruzi, and the promastigote forms of L. donovani and P. staheli. Some of the compounds reached complete growth inhibition at 1 microg/ml for 48 h of parasite/drug interaction. None of the compounds tested showed significant toxicity against cells of Aedes albopictus, mouse macrophages J-774A.1 and Lycopersicum esculentum at dosages five times greater than used against parasites.

Biochemical and ultrastructural alterations caused by newly synthesized 1,2,4-triazole[1,5a]pyrimidine derivatives against Phytomonas staheli (Trypanosomatidae).

Six compounds, all newly synthesized triazole-pyrimidine derivatives that proved inhibitory of in in vitro growth of epimastigotes in Trypanosoma cruzi and of promastigotes of Leishmania donovani and Phytomonas staheli, were studied to investigate their toxic effects. As a biological model, the plant trypanosome P. staheli, which causes sudden wilt in the oil palm and Hartrot in the coconut palm, was used. The six compounds markedly inhibited macromolecule synthesis (nucleic acids and proteins) by the parasite. The cells treated with these compounds present severe damage in their ultrastructure-intense 'vacuolization, and appearance of lysosomes as well as other residual bodies. The mitochondrial section appeared larger in size. with a swollen matrix. In addition, these compounds changed the excretion of end metabolites, primarily affecting ethanol and acetate excretion, possibly by directly influencing certain enzymes (alcohol dehydrogenase and acetate synthetase) or their synthesis. 2000 Elsevier Science Ltd.

Inhibition of cryptosporidium parvum in vitro by 9-(alkylthio)acridine derivatives.

The synthesis of several acridinic thioethers is described. Compounds prepared were tested in vitro as potential drugs against the opportunistic infection known as cryptosporidiosis. With a view to predict activity, the quantitative structure-activity relationships were investigated. Correlations between experimental data and either log P or pKa are discussed.