Lectin receptors distribution in the surface membrane of Trypanosoma cruzi blood forms collected from mice submitted to specific treatment

The author investigated the distribution of lectin receptors on Trypanosoma cruzi blood forms collected from mice inoculated with, respectively, the drug-resistant and drug-sensitive strains VL-10 and CL, and treated with the two standard active nitroheterocyclic compounds nifurtimox and benznidazole used for treatment of human Chagas' disease. Blood trypomastigotes purified in Fycoll-Hypaque were incubated with fluorescein-labelled lectins Con A, WGA, EE, WFA, TPA and PNA and then microscopically examined. Neither qualitative or quantitative differences in the fluorescence intensity could be detected between parasites from VL-10 and CL strains submitted or not to treatment. The results suggest that both strains do not differ in their surface membrane carbohydrate moieties. Moreover, the rapid clearance of blood forms the drug-sensitive strain in animals treated with singlo doses of both compounds is not likely to depend on membrane alterations expressed by changes in the carbohydrate components. furthermore, resistance or sensitivity to drugs is not apparently related to carbohydrate distribution on T. cruzi blood forms.

Advances in pharmacological studies of silymarin

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/Kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.5% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-0-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular systen have been studied in this university since 1980. O. O silymarin 800 mg/Kg/d or silybin 600 mg/Kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipenic rats. Further studies showed that silymarin enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis. The results showed silymarin 80 mg or silybin 60 mg decreased in vitro platelet aggregation (porcentagem) in rats. The maximal platelet aggregation induced by ADP declined significantly, and time to reach maximal platelet aggregation and five-minute disaggregation didn't change. In our experiments, iv silybin 22,4 mg/kg lowered the amplitude and duration of diastolic blood pressure (DBP) more than those of systolic (SBP), but the descending aortic blood flow, cardiac contractility and ECG did not change significantly in anesthetized open-chest cats. The results indicated a reduction of peripheral resistance and dilatatory action on the resistant blood vessels. These effects are beneficial to coronary heart disease. We also observed the effects of silybin on morphological change, the release of glutamic oxaloacetate aminotrasferase (GOT) and lactate dehydrogenase (LDH) as well as the radioactivity of 3H-TdR incorporated into DNA in normal cardiac cells and cells infected by coxsackie B5, virus os newborn rats. The results showed that silynin did not affect the morphology of normal cell, and that the pathological change of cells infected by virus was delayed and reduced as compared to control. We have investigated the effect of silybin on synthesis and release of LTs in the cultured porcine cerebral basilar arteries (PCBA). Silybin 100 and 500 µmol/L declined the amounts of LTs released from the PCBA incubsated in the presence of A 23187, AA and indomenthacin. The result suggests that silybin can inhibit the activity of 5-lipoxygenase of cerebral blood vessel and may protect the brain from ischemia.

Risk factors for Trypanosoma cruzi infection among blood donors in Central Brazil

Characteristics and possible risk factors associated with Trypanosoma cruzi infection among blood donors were assessed within a routine screening programme in blood banks in an endemic area of Chagas disease. 6,172 voluntary blood donors were interviewed and tested for anti-T. cruzi antibodies by Haemagglutination and Complement Fixation tests in six blood banks in Goiânia-Central Brazil from October 1988 to April 1989. An overall prevalence of 2.3% for T. cruzi infection was obtained, being 3.3% for first-time blood donors, and 1.9% for regular ones (p < 0.01). Considering this seropositivity among regular blood donors, selection of candidates relying only on the history of previous donation was found to be inadequate. The risk of infection increased inversely with the degrees of education and monthly income. There was a 9.2 risk of infection (95% CI 3.8-22.6) for those who had lived more than 21 years in an endemic area compared to subjects who had never lived in rural settings, after multivariate analysis. These informations may help to review the criteria of selection of donors in order to improve quality of blood products in endemic areas.

Evaluation of some organic compounds on bloodstream forms of Trypanosoma cruzi

Accidental transmission of Chagas' disease to man by blood transfusion is a serious problem in Latin-America. This paper describes the testing of several synthetic, semi-synthetic, and natural compounds for their activity against blood trypomastigotes in vitro at 4-C. The compounds embody several types of chemical structures: benzoquinone, naphthoquinone, anthracenequinone, phenanthrenequinone, imidazole, piperazine, quinoline, xanthene, and simple benzenic and naphthalenic derivates. Some of them are for the first time tested against Trypanosoma cruzi. The toxic effect these compounds on this parasite was done by two quite distinct sets of experiments. In one set, the compounds were added to infected blood as ethanolic solution. In this situation the most active one was a furan-1, 2-naphthoquinone, in the same range as gentian violet, a new fact to be considered in the assessment of structure-activity relationships in this class of compounds. In other set, we tentatively evaluated the biological activity of water insoluble compounds by adding them in a pure form without solvent into infected blood. In this way some appear to be very active and it was postulated that the effectiveness of such compounds must result from interactions between them and specific blood components.

Temperature effect upon blood consumption in Triatoma infestans

Different blood consumption speed was observed in Triatoma infestans - nymphs and adults - exposed to 12 degrees C and 28 degrees C. Exposure to optimal temperature (28 degrees C) allows the insects to consume blood at a rate of 9% per day. Significative relationship between blood amount present in the promesenteron and consumed blood was found at 28 degrees. Consumption of blood was drastically reduced at the lowest temperature. Accordingly, lack of ovaric development, oviposition and mating behaviour was observed in insects kept at 12 degrees C. Relationship between laboratory and field observations are discussed.

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.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Mechanisms of protective immunity against asexual blood stages of Plasmodium falciparum in the experimental host Saimiri

In the Saimiri monkey, an experimental host for human malaria, acquired protection against Plasmodium falciparum blood stages depends on the IgG antibody populations developed. In vivo protective anti-falciparum activity of IgG antibodies is correlated with the in vivo opsonizing activity promoting phagocytosis of parasited red bloood cells. In contrast, non protective antibodies inhibit this mechanism by competing at the target level. A similar phenomenon can be and human infection. Anti-cytoadherent and anti-rosette antibodies developed by Saimiri and humans prevent the development of physiopathological events like cerebral malaria which can also occur in this experimental host. Furthermore, transfer to protective human anti-falciparum IgG antibodies into infected Saimiri monkeys exerts an anti parasite activity as efficient as that observed when it is transfered into acute falciparum malaria patients, making the Saimiri an even more attractive host. Studies on the role of immunocompetent cells in the protective immune reponse are still in their infancy, however the existance of a restricted polymorphism of MHC II class molecules in the Saimiri confers additional theoretical and practical importance to this model.

Modern immunological approaches to assess malaria transmission and immunity and to diagnose plasmodial infection

The present paper reviews our recent data concerning the use of immunological methods employing monoclonal antibodies and synthetic peptides to study malaria transmission and immunity and to diagnose plasmodial infection. As concerns malaria transmission, we studied the main vectors of human malaria and the plasmodial species transmitted in endemic areas of Rondônia state, Brazil. The natural infection on anopheline was evaluated by immunoradiometric assay (IRMA) using monoclonal antibodies to an immunodominant sporozoite surface antigen (CS protein) demonstrated to be species specific. Our results showed that among six species of Anopheles found infected, An. darlingi was the main vector transmitting Plasmodium falciparum and P. vivax malaria in the immediate vicinity of houses. In order to assess the level of anti-CS antibodies we studied, by IRMA using the synthetic peptide corresponding to the repetitive epitope of the sporozoite CS protein, sera of individuals living in the same areas where the entomological survey has been performed. In this assay the prevalence of anti-CS antibodies was very low and did not reflect the malaria transmission rate in the studied areas. In relation to malaria diagnosis, a monoclonal antibody specific to an epitope of a 50 kDa exoantigen, the major component of supernatant collected at the time of schizont rupture, was used as a probe for the detection of P. falciparum antigens. This assay seemed to be more sensitive than parasitological examination for malaria diagnosis since it was able to detect plasmodial antigens in both symptomatic and asymtomatic individuals with negative thick blood smear at different intervals after a last parasitologically confirmed confirmed attack of malaria.

Mechanisms of immune protection in the asexual blood stage infection by Plasmodium falciparum: analysis by in vitro and ex-vivo assays

Mechanisms of immune protection against the asexual blood stage infection by Plasmodium falciparum are reviewed. Recent studies of two independent lines of research developed at the Institute Pasteur, in humans and primate infections clearly indicate an obligatory interaction of antibodies and effector cells to express the anti-parasitic effect.