Synthesis and in vitro activity of new biguanide-containing dendrimers on pathogenic isolates of Acanthamoeba polyphaga and Acanthamoeba griffini.

The genus Acanthamoeba can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage that many times causes infection relapses. In an attempt to add new agents to our chemotherapeutic arsenal against acanthamebiasis, two Acanthamoeba isolates were treated in vitro with newly synthesized biguanide dendrimers. Trophozoite viability analysis and ultrastructural studies showed that dendrimers prevent encystment by lysing the cellular membrane of the amoeba. Moreover, one of the dendrimers showed low toxicity when tested on mammalian cell cultures, which suggest that it might be eventually used as an amoebicidal drug or as a disinfection compound in contact lens solutions.

Molecular epidemiology of parasitic protozoa and Ehrlichia canis in wildlife in Madrid (central Spain).

Wildlife species are involved in the transmission of diverse pathogens. This study aimed to monitor raccoons (Procyon lotor), American minks (Neovison vison), and red foxes (Vulpes vulpes) as potential reservoirs in central Spain. Specifically, 200 spleen and fecal samples (from 194 raccoons, 3 minks, and 3 foxes) were analyzed molecularly by PCR/qPCR and sequencing for the presence of piroplasmids, Hepatozoon spp., Toxoplasma gondii, and Ehrlichia canis infections in the Community of Madrid (Spain). Biological samples were obtained in the years 2014, 2015, and 2016. No pathogen DNA was found in fecal samples. In contrast, analysis of raccoon spleen samples revealed that Toxoplasma was the most prevalent pathogen (prevalence 3.6 ± 2.6%), followed by Hepatozoon canis and E. canis (each with a prevalence of 2.57 ± 2.2%). Hepatozoon canis was also diagnosed in all three of the analyzed foxes. Analysis of yearly prevalence showed that tick-borne pathogens were less frequent in raccoon in 2015, a dry and warm year compared both to 2014 and 2016. These data suggest that fecal PCR assays are unsuitable for detection of DNA of non-erythrocytic pathogens. Furthermore, they demonstrate that the raccoon (an invasive species often living in proximity to domestic areas) and the red fox are putative reservoirs for pathogenic organisms in the Community of Madrid.

A survey for gregarines (Protozoa: Apicomplexa) in arthropods in Spain.

Gregarines thrive in the digestive tract of arthropods and may be deleterious to their hosts, especially when present in high densities. The impact of parasites on these invertebrates may affect both the ecosystem equilibrium and human economic activities. However, information available on gregarines in Spain is limited. Therefore, a microscopic study on prevalence of gregarine infection in 560 insects and crustaceans was undertaken in Madrid and Tarragona.Gregarina ormierei (78 % prevalence), Stylocephalus gigas (56 %), Oocephalus hispanus (13 %) and Actinocephalus permagnus (only one infected out of six beetles examined) were found in coleopteran hosts. Gregarina ovata and G. chelidurellae showed moderate frequency of infection (35 %) in dermapterans. An undescribed Gregarina sp. (76 % prevalence) was observed for the first time in freshwater decapod crustaceans. Interestingly, G. ormierei showed a noticeable phenotypic dimorphism, which justifies its redescription based on modern taxonomic criteria. Sequences of the 18S rRNA gene could be obtained only in the presence of highly prevalent gregarines. G. ormierei and Gregarina sp. were related (85 and 94 % identity by BLASTN, respectively) to G. basiconstrictonea and G. cloptoni, respectively, whereas S. gigas was closely related to both Xiphocephalus ellisi and S. giganteus (>97 % identity). Phylogenetic trees based on ribosomal sequences unequivocally grouped these new isolates either with the Gregarinidae (G. ormierei and Gregarina sp.) or the Stylocephalidae (S. gigas).

Characterization of a human-pathogenic Acanthamoeba griffini isolated from a contact lens-wearing keratitis patient in Spain.

Amoebae were isolated from contact lenses of a symptomatic lens wearer in Spain. Protozoa were characterized by studying their morphology, biology, protease activity and the 18S rRNA gene sequence. Morphology of the organism was observed by light microscopy, scanning electron microscopy and transmission electron microscopy. Its structure corresponded to an amphizoic amoeba. The protozoa grew well at 37 °C and poorly at lower temperatures. In addition, it was capable of lysing mammalian cells in vitro. A major 56 kDa proteolytic enzyme was observed in amoeba crude extracts by gelatin-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Most proteolytic enzymes in protozoa extracts showed significant activity over a wide range of pH (3-9) and temperature (8-45 °C) values. The assays on inhibition of protease activity indicated strongly that enzymes detected in amoeba extracts corresponded to serine proteases and, to a lesser extent, cysteine proteases. The use of proteinase inhibitors on a tissue culture model proved that the proteinase activity is critical for developing focal lesions in HeLa cell monolayers. Finally, partial sequencing of the 18S ribosomal RNA gene and phylogenetic analyses indicated that the isolate is closely related to Acanthamoeba griffini H37 from the UK (T3 genotype).

Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.

A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism.

In vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against Acanthamoeba castellanii UAH-T17c3 trophozoites.

Acanthamoeba is one of the most common free-living amoebas which is widespread in the environment and can infect humans, causing diseases such as keratitis and encephalitis. In this paper we examine for the first time the amebicidal activity of the family of cationic dendrimers nG-[Si{(CH(2))(3)N(+)(Me)(Et)(CH(2))(2)NMe(3) (+)}2I(-)]( x ) (where n denotes the generations: zero (n = 0, x = 1), first (n = 1, x = 4), and second (n = 2, x = 8); for simplicity, they were named as 0G-CNN2, 1G-CNN8, and 2G-CNN16, respectively) against Acanthamoeba castellanii UAH-T17c3 trophozoites. In order to test the amebicidal activity, we cultured the strain A. castellanii UAH-T17c3 in PYG-Bactocasitone medium and later, we treated it with different concentrations of these dendrimers and monitored the effects and damage by optical count, flow cytometry, and scanning electron microscopy. The results showed that all the nanosystems assayed had a strong amebicidal activity. The dendrimer 1G-CNN8 was the most effective against the amoeba. In the morphology of treated throphozoites of A. castellanii UAH-T17c3 analyzed by light and scanning electron microscopy techniques, morphological changes were evident in amoeba cells, such as loss of pseudopodia, ectoplasm increase, roundness, and cellular lysis. Furthermore, flow cytometry results showed alterations in cell granularity, which was dose-time dependent. In conclusion, this family of cationic carbosilane dendrimers has a strong amebicidal activity against the trophozoites of A. castellanii UAH-T17c3 in vitro. They could potentially become new agents significant to the development of new amebicidal compounds for prevention and therapy of Acanthamoeba infections.

In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites.

The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

Acanthamoeba castellanii: in vitro UAH-T17c3 trophozoite growth study in different culture media.

Acanthamoeba is one of the most common free-living amoebae. It is widespread in the environment and can infect humans, causing diseases such as keratitis and encephalitis. In this study, we used a strain of Acanthamoeba castellanii (UAH-T17c3) isolated from cooling towers, and we evaluated the efficiency of three different culture media in its growth, with the aim of selecting one which allowed better growth, was easier to prepare, and was able to keep the trophozoites by long periods of time. We compared the growth of A. castellanii in peptone-yeast extract-glucose (PYG, the most commonly used medium to grow this strain) to the growth in PYG-Bactocasitone (PYG with 2% Bactocasitone) and brain-heart infusion broth (BHI is a standard microbiological medium rarely used in the culture of amoebae). Flow cytometry and cell count results showed all three media allowed the growth of trophozoites. PYG-Bactocasitone was shown to be the best for long-term culture. The BHI and PYG-Bactocasitone media have not been used for Acanthamoeba spp. trophozoite growth. In view of the results, we can affirm that these media are adequate to grow the above-mentioned strain for in vitro screening assays.

Amoebicidal activity of cationic carbosilane dendrons derived with 4-phenylbutyric acid against Acanthamoeba griffini and Acanthamoeba polyphaga trophozoites and cysts.

Amoebae from the genus Acanthamoeba are important pathogens responsible for severe illnesses in humans such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. In the last few decades, AK diagnoses have steadily increased. Most patients suffering from AK were contact lens users and the infection was related to poor hygiene. However, therapy is not yet well established, and treatments may last for several months due to resistance. Moreover, these treatments have been described to generate cytotoxicity. Therefore, there is an urgent need to develop new therapeutic strategies against AK. In this study, the amoebicidal activity of different generation cationic carbosilane dendrons derived with 4-phenylbutyric acid was demonstrated against Acanthamoeba polyphaga and Acanthamoeba griffini trophozoites and cysts. In addition, the combination of chlorhexidine digluconate and the most effective dendron (ArCO2G2(SNMe3I)4) showed an in vitro effect against Acanthamoeba trophozoites and cysts, reducing the minimal trophozoite amoebicidal concentration as well as concentrations with cysticidal activity.

Isolation and molecular characterization of Acanthamoeba from patients with keratitis in Spain.

In order to improve our knowledge on the epidemiology of amoebic keratitis, as well as the identification of Acanthamoeba isolates, we have isolated Acanthamoeba spp. from five symptomatic patients in Spain in the present study. All isolates were grown in axenic liquid medium, with only one exception. The morphology of these isolates were characterized by optical and scanning electron microscopy. Their structural features corresponded to those of amphizoic amoebae (namely Acanthamoeba spp.). The molecular characterization of the five Acanthamoeba isolates yielded six sequences. Almost complete 18S rRNA gene sequences (>2000bp) were obtained from three isolates and partial sequences (∼1500bp) from the other two. A robust phylogenetic analysis based on the almost complete 18S rRNA sequence showed that four isolates belonged to the T4 genotype and the other one to the T3 genotype. However, all isolates were identified as T4 genotype using the ASA.S1 fragment. As previously suggested by other researchers, only a robust phylogenetic approach may be helpful in identifying Acanthamoeba genotypes. In addition, new data on the phylogenetic relationships among the Acanthamoeba genotypes is provided and discussed.

[Evoked potentials in a population of children below 1,500 grams at birth: a description and probabilities]. / Potenciales evocados en una población de niños menores de 1.500 gramos: descripción y probabilidades.

INTRODUCTION: Bioelectrical behaviour was studied in a group of low birth weight children. AIM: To evaluate whether the characteristics of the waves of the brain potentials in these children, who weighed less than 1500 g at birth and experienced anomalous circumstances and events during their perinatal period, would help reach an early diagnosis of the possible developmental disorders they might suffer later on in life. SUBJECTS AND METHODS: Both visual and auditory cerebral evoked potentials were recorded in a group of children born underweight and the results were compared with the findings from another group of healthy children who were born in normal physiological conditions and were apparently free of any kind of pathology. RESULTS: In the waves and locations that were examined, the problem group displayed latencies that were longer than those of the control group; in contrast, no statistically significant differences were found in the amplitude, regardless of the location. Low gestational age and lower weight made latencies longer, but no relationship was found between latencies and the other perinatal features that were studied. CONCLUSIONS: Children with low weight at birth have slower wave latencies than normal children. This slowing, which is inversely proportional to the weight and weeks of gestation, is considered to be an anomalous sign that could be related to brain immaturity, delayed development or to disorders affecting myelination. Moreover, the amplitude, which has received far less attention from researchers, is usually shorter in these processes, although in our study we found no differences with the group of healthy children--only very slightly in the P300, in the weeks and the weight, and the N100 only in one location with respect to weight. Since these children usually have developmental disorders, the use of evoked potentials could be a very useful tool in their detection and ensuing therapy.

In vitro anti-Acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms.

Acanthamoeba sp. are the causative agents of severe illnesses in humans such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Medical therapy is not yet well established. Treatments of AK last for several months and generate toxicity, resistances appear due to the cysts stage and recurrences can occur. In this study has been demonstrated that the combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties has in vitro synergistic effect against Acanthamoeba polyphaga. This synergy provokes an important reduction in the minimal trophozoite amoebicidal concentration (MTAC) of CLX, which means a reduction of their toxic effects on human cells. Moreover, some CLX/dendrimer combinations show important activity against the cyst resistance stage.

The effects of albendazole and albendazole sulphoxide combination-therapy on Echinococcus granulosus in vitro.

Protoscoleces of Echinococcus granulosus were incubated in vitro with decreasing concentrations of either albendazole (ABZ) or albendazole sulphoxide (ABZ.SO) (50, 10, 1 and 0.1 micrograms ml-1), and in combination. Viability was assessed by the methylene blue exclusion test and establishment of infection in mice. Protoscolex ultrastructure was determined by means of transmission and scanning electron microscopy. ABZ and ABZ.SO, when used separately had protoscolicidal activity after a longer incubation period (30 days) than when used as combined compounds. When incubated in the presence of ABZ + ABZ.SO, protoscolex viability dropped rapidly. That is, protoscoleces were all non-viable at 12 days of exposure, with no cyst developing following their inoculation into mice. The ultrastructural changes induced by ABZ or ABZ.SO alone, were: (a) rostellar disorganization, (b) formation of numerous blebs on the tegument, (c) loss of the microtriches, (d) increased vesiculation within the tegumentary cytons together, (e) an increase in lipid deposits and (f) depletion of glycogen reserves. After incubation with combined ABZ and ABZ.SO the tegument contained numerous blebs which became detached, leaving debris only, some intact nuclei being discernible in the protoscolex parenchyma.

In vitro stress response to elevated temperature, hydrogen peroxide and mebendazole in Trichinella spiralis muscle larvae.

Three stimuli, elevated temperature, hydrogen peroxide and mebendazole, were compared for their ability to induce heat-shock responses in Trichinella spiralis muscle larvae (L1). In vitro effectiveness of each 'stressor' was evaluated by viability score, protein content and levels of hsp90, hsp70 and hsp60. Detection of the respective heat-shock proteins was done by Western blotting and the heat-shock proteins and quantitation of the immunoblots by image analysis. Exposure of L1 to elevated temperature (e.g. 45 degrees C, 2 h) had no measurable effect. However, exposure to hydrogen peroxide resulted in the induction of constitutive and higher mol. wt heat-shock proteins. In these experiments, heat-shock protein induction correlated strongly with other damage parameters, including loss of viability and increased mortality. Larvae stored in the presence of mebendazole showed no signs of damage. These data indicate that when L1 suffer damage through the action of stimuli, enhancement of heat-shock protein production and damage suffered are causally related.

Development of chemotherapeutic model for the in vitro screening of drugs against Echinoccus granulosus cysts: the effects of an albendazole-albendazole sulphoxide combination.

This paper describes a novel experimental model for the screening of putative drugs against the metacestode stage of E. granulosus using hydatid cysts derived from in vitro culture of protoscoleces. The effects of an ABZ+ABZ.SO combination against cultured and murine cysts were studied with this in vitro model system. This treatment produced loss of turgidity of the cultured cysts in less time than in the murine cysts but the ultrastructural tissue damage observed in both cultured and murine cysts was similar. The ultrastructural changes induced by ABZ+ABZ.SO were: (i) vacuolation of the distal cytoplasm that extended to the tegumentary cells of the germinal membrane; (ii) increased number of mitochondria; (iii) partial loss of microtriches; (iv) increased number of autophagosomes; and (v) an increase in lipid deposits.

In vivo effect of oral albendazole and albendazole sulphoxide on development of secondary echinococcosis in mice.

This paper describes the effectiveness of ABZ, ABZ.SO and ABZ + ABZ.SO treatment in mice infected with Echinococcus granulosus protoscoleces. The results were evaluated in two ways: measuring the number and wet weight of developed cysts and by transmission electron microscopy (TEM) of tissue cyss. ABZ and ABZ + ABZ.SO had an important effect upon larval growth in E. granulosus. The ultrastructural changes noted were: vacuolation of tegumentary cells of the germinal membrane; increased number of mitochondria; increased number of autophagosomes and an increase in lipid deposits.

In vitro shock response to different stressors in free living and pathogenic Acanthamoeba.

Three stresses, viz heat, oxidative and pH shocks, were applied to cultures of three species of Acanthamoeba, free-living Acanthamoeba rhysodes and pathogenic Acanthamoeba castellanii and Acanthamoeba culbertsoni. The effect of each stressor on trophozoite integrity was evaluated by the amount of heat shock protein (HSP)60 and HSP70 produced and by exclusion of 0.2% Congo Red. HSP60 and HSP70 levels were estimated using Western blotting and subsequent densitometric analyses. Unstimulated trophozoites from A. rhysodes produced the lowest background levels of HSP60 and HSP70 and were the amoebae most affected by (mammalian-type) stresses as judged by their enhanced HSP production and decreased viability upon exposure to such conditions. In contrast, unstimulated Acanthamoeba of the pathogenic variety had relatively high background levels of test HSPs and seemed undisturbed by the types of stresses they must deal with when entering their hosts. These studies suggest that high HSP levels in amphizoic acanthamoebae may indicate their involvement in (i) tolerance induction to hosts' stressors and/or (ii) in species' virulence.

Viability of Echinococcus granulosus cysts in mice following cultivation in vitro.

The viability of hydatid cysts developed in vitro for 90 days was assessed by implantation into mice. Cysts removed from mice at 270 days post-infection (p.i.) increased their size 13.5-fold and contained several brood capsules containing protoscoleces. Thus, cysts remain viable after prolonged in vitro culture. The implantation in mice of 15 cysts developed in vitro yielded an average of 10 cysts per mouse, which is indicative of a high survival rate in these experimental infections. The ultrastructural study of cysts recovered from mice 270 days p.i. showed that the germinal membrane was more compact than before implantation and several layers of tegumental cells had developed. Observations of cysts removed from mice indicated that the plasma membrane surrounding microtriches had prolongations opening into the laminated layer.

Using heat shock proteins as indicators of the immune function in wistar rats during a secondary Trichinella spiralis infection.

The muscle, liver, brain and spleen tissues from Wistar rats with either a primary Trichinella spiralis infection alone, or reinfected 45 days after primary infection, were collected at Days 1, 7, 14, 20 and 27 post reinfection. They were then assayed for levels of four heat shock proteins (HSPs), i.e. hsp90, hsp70, hsp60 and hsp25. Detection and quantitation of the separate HSPs in tissue specimens were achieved using Western blot and image analysis technique, respectively. The results show that the elements affecting altered expression of rat organs' HSP were 'neutralized' by resistance-related events in immunized rats. Thus, while rat organs exhibited varying HSP levels in primary infections [Martinez, J., Perez-Serrano, J., Bernadina, W., Rodriguez-Caabeiro, F., 1999. Parasitology 118, 202-209], there was, in reinfected versus primary-infected rats, no difference in test HSPs levels in any organ, and at any time within the time course of this study. We have interpreted the results by using the model that involves induction of anti-T.spiralis immunity during primary infection and (almost) complete removal of effectors of tissue injury (infective T.spiralis larvae and newborn larvae) during reinfection.

Detection of heat shock protein-70 from Trichinella spiralis larvae using a modification of the routine western blotting procedure.

This is the first study that establishes a standardized western blotting method for the detection of heat shock protein (HSP) 70 from Trichinella spiralis using (selected) monoclonal antibodies (mAbs). Enhancement of HSP transfer onto the supportive membrane and increased retention of protein by the membrane are prominent features of the procedure. The reactivity of T. spiralis HSP70 on western blots was substantially increased by the use of a 10% acrylamide gel, the optimization of conditions during electrotransfer, and transfer onto Immobilon membranes. These data indicate that mAbs actually capable of detecting the agent of interest may be discarded because of nonoptimal testing conditions. We suggest that this method will aid in understanding the role and function of T. spiralis HSP70 in host-parasite (inter)relationship(s).