In vitro screening of Amazonian plants for hemolytic activity and inhibition of platelet aggregation in human blood

In the present study, different aerial parts from twelve Amazonian plant species found in the National Institute for Amazon Research's (INPA's) Adolpho Ducke Forest Reserve (in Manaus, Amazonas, Brazil) were collected. Separate portions of dried, ground plant materials were extracted with water (by infusion), methanol and chloroform (by continuous liquid-solid extraction) and solvents were removed first by rotary evaporation, and finally by freeze-drying which yielded a total of seventy-one freeze-dried extracts for evaluation. These extracts were evaluated initially at concentrations of 500 and 100 µg/mL for in vitro hemolytic activity and in vitro inhibition of platelet aggregation in human blood, respectively. Sixteen extracts (23 % of all extracts tested, 42 % of all plant species), representing the following plants: Chaunochiton kappleri (Olacaceae), Diclinanona calycina (Annonaceae), Paypayrola grandiflora (Violaceae), Pleurisanthes parviflora (Icacinaceae), Sarcaulus brasiliensis (Sapotaceae), exhibited significant inhibitory activity towards human platelet aggregation. A group of extracts with antiplatelet aggregation activity having no in vitro hemolytic activity has therefore been identified. Three extracts (4 %), all derived from Elaeoluma nuda (Sapotaceae), exhibited hemolytic activity. None of the plant species in this study has known use in traditional medicine. So, these data serve as a baseline or minimum of antiplatelet and hemolytic activities (and potential usefulness) of non-medicinal plants from the Amazon forest. Finally, in general, these are the first data on hemolytic and inhibitory activity on platelet aggregation for the genera which these plant species represent.

Use of glass beads and CF 11 cellulose for removal of leukocytes from malaria-infected human blood in field settings

Passage of malaria infected blood through a two-layered column composed of acid-washed glass beads and CF 11 cellulose removes white cells from parasitized blood. However, because use of glass beads and CF 11 cellulose requires filtration of infected blood separately through these two resins and the addition of ADP, the procedure is time-consuming and may be inapropriate for use in the field, especially when large numbers of blood samples are to be treated. Our modification of this process yields parasitized cells free of contaminating leukocytes, and because of its operational simplicity, large numbers of blood samples can be processed. Our procedure also compares well with those using expensive commercial Sepacell resins in its ability to separate leukocytes from whole blood. As a test of usefulness in molecular biologic investigations, the parasites obtained from the blood of malaria-infected patients using the modified procedure yield genomic DNA whose single copy gene, the circumsporozite gene, efficiently amplifies by polymerase chain reaction.

Identification of the Boudicca and Sinbad retrotransposons in the genome of the human blood fluke Schistosoma haematobium

Schistosomes have a comparatively large genome, estimated for Schistosoma mansoni to be about 270 megabase pairs (haploid genome). Recent findings have shown that mobile genetic elements constitute significant proportions of the genomes of S. mansoni and S. japonicum. Much less information is available on the genome of the third major human schistosome, S. haematobium. In order to investigate the possible evolutionary origins of the S. mansoni long terminal repeat retrotransposons Boudicca and Sinbad, several genomes were searched by Southern blot for the presence of these retrotransposons. These included three species of schistosomes, S. mansoni, S. japonicum, and S. haematobium, and three related platyhelminth genomes, the liver flukes Fasciola hepatica and Fascioloides magna and the planarian, Dugesia dorotocephala. In addition, Homo sapiens and three snail host genomes, Biomphalaria glabrata, Oncomelania hupensis, and Bulinus truncatus, were examined for possible indications of a horizontal origin for these retrotransposons. Southern hybridization analysis indicated that both Boudicca and Sinbad were present in the genome of S. haematobium. Furthermore, low stringency Southern hybridization analyses suggested that a Boudicca-like retrotransposon was present in the genome of B. truncatus, the snail host of S. haematobium.

Aluminum induces lipid peroxidation and aggregation of human blood platelets

Aluminum (Al3+) intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL) of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM) stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA) (100 µM) and n-propyl gallate (NPG) (100 µM), inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA) (100 µM), an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast

The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an endothelium. It can be surmised that as occurs in endothelial injury the subcellular components of the syncytiotrophoblast may have pro-or antiaggregatory activity. During congenital Chagas' disease which is associated to trophoblast lesions, platelets may play a role in the development of T. cruzi-induced placentitis. In the present work the aggregatory behaviour of normal human blood platelets was recorded after their challenging with subcellular fractions of syncytiotrophoblast isolated from normal and chagasic women. Nuclear, Mitochondrial, Microsomal and Supernatant fractions isolated from normal and chagasic syncytiotrophoblast failed to induce per se any aggregatory reaction on platelets. When samples of platelet-rich plasma (PRP) were preincubated with normal and chagasic nuclear fractions and then stimulated with collagen at threshold level (CT-PRP) an inhibition of the aggregatory response was observed. Treatment of CT-PRP with normal and chagasic mitochondrial fractions induced inhibition of platelet aggregation whereas only chagasic fraction reduced latency time. Microsornal fraction from normal placentas showed no significant effects on platelet aggregation. It is concluded that subcellular fractions of normal human syncytiotrophoblast do not exhibit any effect on platelet aggregation, whereas those subcellular fractions enriched in intracellular membrane components isolated from chagasic placentas inhibit platelet aggregation.

Haemolytic activity of Actinobacillus actinomycetemcomitans strains on different blood types

Haemolytic activity of sixty nine Actinobacillus actinomycetemcomitans strains on different animal and human blood types was examined by using a trypticase soy agar supplemented with yeast extract (0.5%). Blood types used were: rabbit, sheep and human (A, Rh+; A, Rh-; B, Rh+; B, Rh-; O, Rh+; O, Rh-; AB, Rh+; AB, Rh- groups). Plates were inoculated and, incubated in microaerophilic conditions, at 37ºC, for 48 h. The haemolytic activity of the tested strains was characterized as alpha-haemolysis. Only two isolates were not haemolytic on all blood types (2.9%), two strains were haemolytic only on human blood (one strain on AB, Rh+ group and another one on A, Rh+ and AB, Rh+ groups). No specificity between haemolysin produced by the tested strains and blood type was observed.

Anopheles (Kerteszia) cruzii (DIPTERA: CULICIDAE) IN PERIDOMICILIARY AREA DURING ASYMPTOMATIC MALARIA TRANSMISSION IN THE ATLANTIC FOREST: MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES INDICATES HUMANS AS PRIMARY INTERMEDIATE HOSTS

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

The FML (Fucose Mannose Ligand) of Leishmania donovani: a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovanii infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0 % in antibody response (p<0.001) and reduction of 85.5 % parasite liver burden (p<0.001) was detected in animals immunized with FML saponin, unrespectivety of the immunization route.

Comparison between precipitin and ELISA tests in the bloodmeal detection of Aedes aegypti (Linnaeus) and Aedes fluviatilis (Lutz) mosquitoes experimentally fed on feline, canine and human hosts

The identification of arthropod bloodmeals is important in many epidemiological studies, as, the understanding of the life cycle of vectors and the patogens they transmit, as well as helping to define arthropods' control strategies. The precipitin test has been used for decades, but ELISA is slowly becoming more popular. To compare the two tests for sensitivity, specificity and accuracy to detect small insect bloodmeals, Aedes aegypti or Ae. fluviatilis mosquitoes were fed either on feline, canine or human hosts. Mosquitoes were frozen at 6, 12, 24, 48 or 72 h after feeding. Precipitin test showed better specificity and accuracy and ELISA test showed higher sensitivity. Better results with both tests were achieved when mosquitoes were frozen within 48 h from feeding.

Effect of untreated bed nets on blood-fed Phlebotomus argentipes in kala-azar endemic foci in Nepal and India

Observational studies in the Indian subcontinent have shown that untreated nets may be protective against visceral leishmaniasis (VL). In this study, we evaluated the effect of untreated nets on the blood feeding rates of Phlebotomus argentipes as well as the human blood index (HBI) in VL endemic villages in India and Nepal. The study had a "before and after intervention" design in 58 households in six clusters. The use of untreated nets reduced the blood feeding rate by 85% (95% CI 76.5-91.1%) and the HBI by 42.2% (95% CI 11.1-62.5%). These results provide circumstantial evidence that untreated nets may provide some degree of personal protection against sand fly bites.

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

Entomological aspects of Chagas' disease transmission in the domestic habitat, Argentina

OBJECTIVE: To study the risk of Trypanosoma cruzi domestic transmission using an entomological index and to explore its relationship with household's characteristics and cultural aspects. METHODS: There were studied 158 households in an endemic area in Argentina. Each household was classified according to an entomological risk indicator (number of risky bites/human). A questionnaire was administered to evaluate risk factors among householders. RESULTS: Infested households showed a wide range of risk values (0 to 5 risky bites/human) with skewed distribution, a high frequency of lower values and few very high risk households. Of all collected Triatoma infestans, 44% had had human blood meals whereas 27% had had dogs or chickens blood meals. Having dogs and birds sharing room with humans increased the risk values. Tidy clean households had contributed significantly to lower risk values as a result of low vector density. The infested households showed a 24.3% correlation between time after insecticide application and the number of vectors. But there was no correlation between the time after insecticide application and T. infestans' infectivity. The statistical analysis showed a high correlation between current values of the entomological risk indicator and Trypanosoma cruzi seroprevalence in children. CONCLUSIONS: The risk of T. cruzi domestic transmission assessed using an entomological index show a correlation with children seroprevalence for Chagas' disease and householders' habits.