Isabela Penna Cerávolo

IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control ofToxoplasma gondii replication in human fibroblasts activated with IFN-γ

The ability of up‐regulatory [recombinant (r) IFN‐γ, rIFN‐β and rTNF‐α] and down‐regulatory (rIL‐4, rIL‐10 and rIL‐13) cytokines to control the expression of indoleamine 2,3‐dioxygenase (INDO) and anti‐Toxoplasma activity in the human fibrosarcoma cell line 2C4 was evaluated. Activation of fibroblasts with rIFN‐γ, rIFN‐β and rTNF‐α resulted in augmentation of INDO expression and activity leading to 40.0, 25.0 and 27.0 % inhibition of tachyzoite growth, respectively. An additive effect was observed when host cells were incubated with rIFN‐γ plus rTNF‐α. With regard to the down‐regulatory cytokines we observed that IL‐4 as well as IL‐13, but not IL‐10, induced significant inhibition of IFN‐γ‐induced control of parasite replication, INDO mRNA expression and tryptophan catabolism. Similarly, IL‐4 but not IL‐10 inhibited the cell surface expression of HLA‐DR and CD2 induced by IFN‐γ. Consistent with these findings we were able to detect by reverse transcription‐PCR the expression of mRNA for different chains of IL‐4 and IL‐13 receptors (IL‐4Rα, IL‐13Rα1 and IL‐13Rα2) but not for IL‐10 receptor in the 2C4 and other human lung fibroblast cell lines (LL24 and MRC5). Together our results indicate that IL‐4 and IL‐13, but not IL‐10, are implicated in the negative regulation of IFN‐γ‐induced anti‐Toxoplasma activity in human cells from fibroblast lineage.

Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy‐positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non‐malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty‐three individuals participated in the five cross‐sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non‐cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine‐rich region II, DBPII), we performed in vitro assays with mammalian cells expressing DBPII sequences which were homologous or not to those from the outbreak isolate. In non‐immune individuals, the results of a 12‐month follow‐up period provided evidence that naturally acquired inhibitory antibodies to DBPII are short‐lived and biased towards a specific allele.

Inhibitory Properties of the Antibody Response to Plasmodium vivax Duffy Binding Protein in an Area with Unstable Malaria Transmission

The function of the Plasmodium vivax Duffy binding protein (DBP) during the erythrocyte invasion process is critical for successful parasite growth and pathogenesis in human infections. Although DBP is the subject of intensive malaria vaccine research, investigations on the functional proprieties of anti‐DBP antibodies in the human population have been limited [Infect Immun68 (2000) 3164]. In the present study, we examined the ability of sera from different populations of the Brazilian Amazon – an area of markedly unstable malaria transmission – to inhibit the erythrocyte‐binding function of the DBP ligand domain (region II, DBPII). We found that long‐term exposure to malaria in the Amazon area elicits DBP‐specific antibodies that inhibit the binding of different DBPII variants to erythrocytes. Despite the great variability of inhibitory antibody responses observed among study participants, we observed a positive correlation between erythrocyte binding‐inhibitory activity and enzyme‐linked immunosorbent assay anti‐DBP antibodies. Of importance, there was a non‐significant tendency towards increased levels of anti‐DBP antibodies among individuals with asymptomatic P. vivax infections.

Characterization of Trypanosoma cruzi strains isolated from chronic chagasic patients, triatomines and opossums naturally infected from the State of Rio Grande do Sul, Brazil

Thirty-five Trypanosoma cruzi strains were isolated from chronic chagasic patients, triatomines and opossums from different municipalities of the State of Rio Grande do Sul. Parasites were characterized by means of mice infectivity, enzyme electrophoresis and randomly amplified polymorphic DNA (RAPD) analysis. Twenty-nine strains were isolated from chagasic patients, 4 from triatomines (2 from Triatoma infestans and 2 from Panstrongylus megistus) and 2 from opossums Didelphis albiventris. Thirty-three T. cruzi strains were of low and 2 strains of high virulence in mice. Both virulent strains were isolated from P. megistus. Isoenzyme analysis of the strains showed 3 different zymodemes. Eleven strains isolated from chagasic patients and 2 from D. albiventris were Z2. Eighteen strains from patients and 2 from T. infestans were ZB and 2 T. cruzi strains isolated from P. megistus were Z1. RAPD profiles obtained with 4 random primers showed a high genetic heterogeneity of the T. cruzi strains. Zymodeme 2 and ZB strains were the more polymorphic. A band sharing analysis of the RAPD profiles of Z2 and ZB strains using 3 primers, showed a very low percentage of shared bands, 20% among 13 ZB strains and 14% among 13 Z2 strains. According to the isoenzyme results, 3 T. cruzi populations were present in State of Rio Grande do Sul. Zymodeme 2 and ZB strains were found infecting man (domiciliar transmission cycle) whereas Z1 strains were found infecting the sylvatic vector P. megistus.

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role in Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey in the Brazilian Amazon Basin. Despite continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the population was reexamined 12 months later. Antibodies from 16 of 50 (36.0%) subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.

New trifluoromethyl triazolopyrimidines as anti-Plasmodium falciparum agents.

According to the World Health Organization, half of the World’s population, approximately 3.3 billion people, is at risk for developing malaria. Nearly 700,000 deaths each year are associated with the disease. Control of the disease in humans still relies on chemotherapy. Drug resistance is a limiting factor, and the search for new drugs is important. We have designed and synthesized new 2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine derivatives based on bioisosteric replacement of functional groups on the anti-malarial compounds mefloquine and amodiaquine. This approach enabled us to investigate the impact of: (i) ring bioisosteric replacement; (ii) a CF3 group substituted at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold and (iii) a range of amines as substituents at the 7-position of the of heterocyclic ring; on in vitro activity against Plasmodium falciparum. According to docking simulations, the synthesized compounds are able to interact with P. falciparum dihydroorotate dehydrogenase (PfDHODH) through strong hydrogen bonds. The presence of a trifluoromethyl group at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine ring led to increased drug activity. Thirteen compounds were found to be active, with IC50 values ranging from 0.023 to 20 µM in the anti-HRP2 and hypoxanthine assays. The selectivity index (SI) of the most active derivatives 5, 8, 11 and 16 was found to vary from 1,003 to 18,478.

Real‐time multiplex allele‐specific polymerase chain reaction for genotyping of the Duffy antigen, the Plasmodium vivax invasion receptor

Background and Objectives  Duffy blood group is of major interest in clinical medicine as it is not only involved in blood‐transfusion risks and occasionally in neonatal haemolytic disease, but it is also the receptor for the human malaria parasite Plasmodium vivax in the erythrocyte invasion. The aim of this study was to develop a rapid and inexpensive approach for high‐throughput Duffy genotyping.

Trypanosoma cruzi : parasitaemia produced in mice does not seem to be related to in vitro parasite-cell interaction

The interaction of Trypanosoma cruzi strains producing subpatent or high parasitaemia in mice with mouse macrophages, Vero and L929 cells was evaluated using tissue culture trypomastigotes. Macrophages were the cells most readily infected while Vero cells presented the highest parasite intracellular multiplication rates. Subpatent strains were equal or more infective than the high parasitaemia. Due to the small number of strains, no correlation could be established between the zymodemes and parasitaemia or parasite-cell interaction in vitro. However parasitaemia in mice does not seem to be related to in vitro parasite-cell interaction.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro.

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Euterpe oleracea pulp extract: Chemical analyses, antibiofilm activity against Staphylococcus aureus, cytotoxicity and interference on the activity of antimicrobial drugs

Euterpe oleracea (Açaí) fruit are widely consumed at the Brazilian Amazon region, and biological potentials such as immunomodulatory and antioxidant have been described for its extracts. However, its antimicrobial properties remain poorly investigated. Here, the antimicrobial and antibiofilm activities of the methanolic extract of an artisanally-manufactured açaí pulp (MEAP) were evaluated against clinical isolates of Staphylococcus aureus. Besides, MEAP interference on the activity of antimicrobial drugs of clinical relevance was explored, and its cytotoxicity against hepatocellular carcinoma cells (HepG2) was investigated. Biochemical and physicochemical properties of the pulp were investigated, and the presence of polyphenols on the extract was confirmed. For the first time, we report that the methanolic extract of açaí pulp is effective against planktonic cells and biofilms of S. aureus, and also decreased the proliferation of HepG2 cells. Statistically significant synergism was observed when the extract was combined to the tested antimicrobials except for erythromycin, and all biochemical and physicochemical parameters ranged within the accepted values established by the Brazilian legislation. Our data open doors for more studies on the antimicrobial activity of phytomolecules isolated from Euterpe oleracea extracts, and also for its combined use with antimicrobial drugs.