ABSTRACT Background: The atypical chemokine receptor 1 (ACKR1) gene encodes the Duffy blood group antigens in two allelic forms: FY*A (FY*01) and FY*B (FY*02), which define the Fy(a+b-), Fy(a-b+), and Fy(a+b+) phenotypes. FY*BES (FY*02N.01) is a single T to C substitution at nucleotide -67 that prevents the FY*B from being expressed in red blood cells (RBCs). Methods: We evaluated 250 residents from a Brazilian malarial endemic region (RsMR). All individuals were phenotyped for Fya and Fyb antigens and genotyped for FY*A, FY*B, FY*B SE , and FY*B weak alleles. Results: Among the 250 individuals, 209 (83.6%) reported previous malaria infection, and 41 (16.4%) did not. The Fy(a+b+) phenotype was present in 97/250 (38.8%), while the Fy(a-b-) was present in 7/250 (2.8%). The FY*A/FY*B was found in 130/250 (52%) and the FY*A/FY*A in 45/250 (18%). The c.1-67>TC was present, in homozygosity, in 11/250 (4.4%). Among 34 individuals with the Fy(a+b-) and FYA*/FYB* mutations, 4/34 (11.8%) had homozygosity for the c.1-67T>C. One individual presented the Fy(a+b-), FY*A/FY*B, and c.1-67T>C in homozygosis, whereas the other presented the Fy(a+b-), FY*A/FY*A, and c.1-67T>C in heterozygosis. Conclusions: We reported a low prevalence of the Fy(a-b-) in persons who had previously been infected with Plasmodium vivax (67.5%). We observed that 102/141 (72.3%) individuals expressing the Fyb antigen had a P. vivax infection, indicating the importance of the Fyb antigen, silenced by a c.1-67T>C mutation in homozygosis, in preventing the P. vivax infection. We showed that the c.1-67T>C mutation in the FY*A did not silence the FY*A expression on RBCs.

A furanoditerpene-enriched fraction was obtained from the fruits of Pterodon emarginatus and submitted to semipreparative high performance liquid chromatography (HPLC). Two new furanoditerpenes, 6α,19β-diacetoxy-7β,14β-dihydroxyvouacapan and 6α-acetoxy-7β,14β-dihydroxyvouacapan, in addition to the known compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate were obtained. Compound structures were determined by 1D and 2D nuclear magnetic resonance (NMR) experiments and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The major compound methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate was evaluated against promastigote forms of Leishmania amazonensis and L. braziliensis, presenting the concentration which causes lysis on 50% of parasites IC50 < 30 µg mL-1.

ABSTRACT This paper reports the in vitro trypanocidal activity evaluation of new carbohydrazide derivatives from 3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine, substituted at C-6 position by phenyl, methyl or trifluoromethyl group. These compounds were evaluated in order to identify the antiparasitic profile against trypomastigote and amastigote forms of Trypanosoma cruzi. The 4-carbohydrazide derivatives presented different profiles of activity. In the investigation of the chemical structure influence in the trypanocidal activity, the results indicated there are large lipophilicity and volume differences among these derivatives. The complementarities of their stereoelectronic and physical-chemical aspects seem to be relevant for the biological activity against T. cruzi.

Sixteen 1,4-diaryl-1,2,3-triazole compounds derived from the natural products veraguensin, grandisin and machilin G were synthesized, with yields of 78-92%. Biological activity tests against Leishmania amazonensis promastigotes showed that three of these compounds were the most active, with maximum inhibitory concentration (IC50) values of 1.1, 3.71 and 7.23 µM. One compound was highly active against Leishmania infantum, with an IC50 value of 5.2 µM, and one derivative showed an IC50 value of 28.6 µM against Trypanosoma cruzi trypomastigotes. Regarding structure-activity relationship (SAR), hybrid 1,2,3-triazolic compounds containing a methylenedioxy group, showed the best antileishmanial and antitrypanosomal activities.

O ácido ursólico é um candidato promissor para o tratamento da doença de Chagas, contudo este fármaco possui baixa solubilidade aquosa e limitada absorção intestinal, ambos os fatores limitantes da biodisponibilidade. Entre as estratégias para potencializar a solubilidade e a dissolução de fármacos lipofílicos, as dispersões sólidas estão crescendo em popularidade. Neste estudo, empregamos mistura dos tensoativos, poloxamer 407 e caprato de sódio, para produzir dispersão sólida contendo ácido ursólico, com o objetivo de aumentar tanto a dissolução do fármaco quanto a atividade tripanocida in vivo. Comparada à mistura física, a dispersão sólida apresentou maior densidade e menor tamanho de partícula. Os resultados da análise de espectroscopia no infravermelho com transformada de Fourier mostraram interações intermoleculares do tipo ligações de hidrogênio entre o fármaco e o poloxamer 407. Os experimentos de difratometria de raio-X revelaram a conversão do fármaco de sua forma cristalina para a forma amorfa, mais solúvel. Consequentemente, a solubilidade do ácido ursólico em dispersão sólida foi aumentada e o fármaco dissolveu-se de maneira mais rápida e completa. Em conjunto com as propriedades promotoras de absorção oral do caprato de sódio, estes resultados explicaram o aumento da atividade tripanocida in vivo do ácido ursólico em dispersão sólida, que também se provou segura após avaliação de citotoxicidade empregando a linhagem celular LLC-MK2.

Ursolic acid is a promising candidate for treatment of Chagas disease; however it has low aqueous solubility and intestinal absorption, which are both limiting factors for bioavailability. Among the strategies to enhance the solubility and dissolution of lipophilic drugs, solid dispersions are growing in popularity. In this study, we employed a mixture of the surfactants poloxamer 407 with sodium caprate to produce a solid dispersion containing ursolic acid aimed at enhancing both drug dissolution and in vivo trypanocidal activity. Compared to the physical mixture, the solid dispersion presented higher bulk density and smaller particle size. Fourier Transform Infrared Spectroscopy results showed hydrogen bonding intermolecular interactions between drug and poloxamer 407. X-ray diffractometry experiments revealed the conversion of the drug from its crystalline form to a more soluble amorphous structure. Consequently, the solubility of ursolic acid in the solid dispersion was increased and the drug dissolved in a fast and complete manner. Taken together with the oral absorption-enhancing property of sodium caprate, these results explained the increase of the in vivo trypanocidal activity of ursolic acid in solid dispersion, which also proved to be safe by cytotoxicity evaluation using the LLC-MK2 cell line.

El objetivo del presente estudio fue evaluar las alteraciones histopatológicas de las placentas provocadas por la infección materna durante la preñez con diferentes cepas de Trypanosoma cruzi. Ratones preñadas, de 60 días de edad, fueron inoculadas por la via intraperitoneal (i.p.) con 2 x 105 tripomastigotes sanguíneos de las cepas colombiana, Y, Bolivia o RC del T. cruzi. Los resultados mostraron que las cepas Bolivia y RC presentan mayor histotropismo por las células placentarias del ratón que las cepas colombiana e Y. El proceso inflamatorio afectó la decidua basal en todos los grupos de ratones infectados, mientras que la cepa colombiana causó intensa degeneración de la decidua basal, con células inflamatorias escasas o ausentes. Las placentas de todos los grupos infectados presentaron áreas de edema, degeneración, necrosis y calcificación, principalmente en la decidua basal. En los animales inoculados con la cepa RC del T. cruzi también fue afectado el trofoblasto de la zona esponjosa. El parasitismo más intenso fue observado en los animales infectados con las cepas Bolivia y RC, envolviendo todas las zonas placentarias, incluso las células trofoblásticas gigantes y el trofoblasto esponjoso. Los resultados permiten concluir que las placentas sufren procesos patológicos diferentes, característicos para cada cepa del T. cruzi.

The objective of the present study was to evaluate the histopathological alterations of the placenta provoked by maternal infection during pregnancy by different Trypanosoma cruzi strains. Pregnant mice, 60 day-old, were i.p. inoculated with 2 x 105 bloodstream trypomastigotes of colombian, Y, Bolivia or RC strains of T. cruzi. The results demonstrated that the Bolivia and RC strains showed higher histotropism for the mouse placental cells than the colombian or Y strains. The inflammatory process in all the infected groups involved the basal decidua, but the colombian strain caused intense decidual degeneration, with rare or absent inflammatory cells. Placentas from all the infected groups displayed aéreas of edema, degeneration, necrosis and calcification, mainly in the basal decidua, but the group infected by the RC strain showed also involvement of the spongiotrophoblast. The highest parasitism was observed with the Bolivia and RC strains, involving all the placenta zones, including the trophoblast giant cells and the spongiotrophoblast. The results show that placentas undergo differentiated pathological processes, characteristic for each strain.

Chagas disease is one of the main public health problems in Latin America. Since the available treatments for this disease are not effective in providing cure, the screening of potential antiprotozoal agents is essential, mainly of those obtained from natural sources. This study aimed to provide an evaluation of the trypanocidal activity of 92 ethanol extracts from species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae against the Y and Bolivia strains of Trypanosoma cruzi. Additionally, cytotoxic activity on LLCMK2 fibroblasts was evaluated. Both the trypanocidal activity and cytotoxicity were evaluated using the MTT method, in the following concentrations: 500, 350, 250, and 100 µg/mL. Benznidazole was used for positive control. The best results among the 92 samples evaluated were obtained with ethanol extracts of Ocotea paranapiacabensis (Am93) and Aegiphila lhotzkiana (Am160). Am93 showed trypanocidal activity against epimastigote forms of the Bolivia strain and was moderately toxic to LLCMK2 cells, its Selectivity Index (SI) being 14.56, while Am160 showed moderate trypanocidal activity against the Bolivia strain and moderate toxicicity, its SI being equal to 1.15. The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from Ocotea paranapiacabensis and Aegiphila lhotzkiana against Chagas disease.

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn b allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn b and KAM+) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.

A atividade de extratos brutos de três espécies de Rapanea (Myrsinaceae) e de Cipadessa fruticosa (Meliaceae) foi avaliada in vitro contra formas tripomastigotas de Trypanosoma cruzi. Foram obtidos 33 extratos de diferentes órgãos das espécies estudadas, sendo que onze deles apresentaram atividades significantes (% de lise > 50) nos ensaios realizados. O fracionamento de um extrato ativo dos galhos de R. lancifolia (99,5%) resultou no isolamento de dois flavonoides (quercetina e taxifolina), que apresentaram baixa atividade tripanocida. De um extrato ativo dos frutos de C. fruticosa (97,7%) foram isolados os limonoides mexicanolídeos cipadesina, mexicanolídeo, febrifugina e cipadesina A, que foram moderadamente ativos sobre T. cruzi. Além disso, outros dois flavonoides (flavona e 7-metoxiflavona), previamente ensaiados contra T. cruzi, foram isolados do extrato hexânico dos galhos de C. fruticosa (100%). Os resultados obtidos aqui sugerem que as plantas avaliadas podem constituir fontes de novas substâncias ativas sobre o T. cruzi.

The activity of crude extracts of three Rapanea species (Myrsinaceae) and Cipadessa fruticosa (Meliaceae) was evaluated in vitro against the trypomastigote forms of Trypanosoma cruzi. Thirty-three extracts from different organs of these species were assayed and eleven of them showed significant activity (lysis % >50). The fractionation of an active extract from branches of R. lancifolia (99.5%) led to the isolation of two flavonoids: quercetin and taxifolin, which have weak trypanocidal activity. Additionally, one active extract from fruits of C. fruticosa (97.7%) afforded mexicanolide limonoids: cipadesin, mexicanolide, febrifugin and cipadesin A, that were slightly active on T. cruzi. Moreover, other two flavonoids (flavone and 7-methoxyflavone), previously assayed against T. cruzi, were isolated from the hexane extract from branches of C. fruticosa (100%). The results presented here suggest that the plants evaluated could be a source of new active compounds against T. cruzi.

Arrabidaea chica (HBK.) Verlot (Bignoniaceae) popularmente, "Pariri", é um arbusto escandente, distribuído do sul do México até a Guiana e Brasil central e é tradicionalmente indicado para tratar sintomas de inflamações e afecções da pele. Seu extrato etanólico foi quimicamente investigado e testado contra leveduras e fungos dermatófitos. A atividade tripanocida do mesmo extrato foi também avaliada. Este trabalho reporta o isolamento de três flavonóides, a inibição total do crescimento de Trichophyton mentagrophytes e um significante efeito tripanocida do extrato etanólico e de suas frações. Não foi detectada qualquer toxicidade aguda relevante, mesmo a uma dose de 1000 mg/kg.

Arrabidaea chica (HBK.) Verlot (Bignoniaceae) vernacular name "Pariri", is a climbing shrub, widespread from South Mexico to Guyana and central Brazil and is traditionally indicated to treat symptoms of inflammations and skin affections. Its ethanol extract was chemically investigated and tested against yeasts and dermatophytic fungi. The trypanocidal activity of the same extract was also evaluated. This work reports the isolation of three flavonoids, the total growth inhibition of Trichophyton mentagrophytes and a significant trypanocidal effect of the ethanol extract and its fractions. No relevant acute toxicity was detected even at a dose of 1000 mg/kg.

The objective of this work was to evaluate karyometrically the alterations caused by different strains of Trypanosoma cruzi in the mouse placenta. Pregnant mice, 60-day oíd, were intraperitoneally inoculated with 2 x 10(5) bloodstream trypomastigotes of the Colombian, Y, Bolivia or RC strain of T cruzi. There were observed clear differences in the karyometric alterations of the trophoblast giant cells and in the spongiotrophoblast cells. The results demónstrate that the Colombian and RC strains cause alterations both in the trophoblast giant cells and in the spongiotrophoblast cells, whereas the Y and Bolivia strains provoke alterations only in the trophoblast giant cells. It is possible concluding that each strain has its own characteristics and that, in spite of the similar type of transmission, it show differential nuances in the placental pathogenic process.

El objetivo de este trabajo fue evaluar cariometricamente las alteraciones causadas por diferentes cepas de T. cruzi en la placenta del ratón. Ratones hembras de 60 días, grávidas, fueron inoculadas, intraperitonealmente, con 2 x 10(5) tripomastigotes sanguíneos de las cepas colombiana, Y, Solivia o RC del T. cruzi. Fueron observadas claras diferencias en las alteraciones cariométricas de las células trofoblásticas gigantes y de las células trofoblásticas de la zona esponjosa. Los resultados demostraron que las cepas colombiana y RC causan alteraciones tanto en las células trofoblásticas gigantes como en las células del trofoblasto esponjoso, mientras que las cepas Y y Bolivia provocan alteraciones solamente en las células trofoblásticas gigantes. Es posible concluir que cada cepa posee características propias y que, a pesar del tipo similar de transmisión, presenta matices diferenciales en el proceso de la patogénesis placentaria.

Analizamos morfológica e histométricamente la glándula sublingual de ratones infectados con la cepa RAL del Trypanosoma cruzi, en machos y hembras. Usamos ratones albinos (Mus musculus), variedad Swiss, inoculados con 2x104 tripomastigotes sanguíneos de la cepa RAL del T. cruzi.. Sacrificamos los animales en el pico de la parasitemia (12º día) y fijamos las glándulas sublinguales en ALFAC. Los cortes histológicos teñidos con HE fueron evaluados histométricamente (cariometría y estereología). La parasitemia fue más elevada en las hembras. Histopatológicamente, los "ácinos" (acini) de los animales infectados eran menores, con escasa secreción, y conductos estriados menores. Los núcleos de las "medialunas" eran menores y había nidos de amastigotes en el citoplasma. Cariométricamente, los núcleos de los ácinos, medialunas y conductos estriados eran menores en los ratones infectados. Estereológicamente, los volúmenes relativos ocupados por ácinos y conductos estriados fueron menores e, inversamente, fue mayor el volumen para el tejido conjuntivo de los machos infectados. Las densidades de superficie de ácinos y conductos fueron mayores, y el diámetro y el espesor de la pared menores, en este grupo. Por otro lado, la densidad de ácinos fue menor, y las de los conductos estriados y tejido conjuntivo, fueron mayores en las hembras infectadas. Las densidades de superficie de ácinos y conductos estriados fueron mayores, mientras que el diámetro y espesor de la pared de los ácinos fueron menores (y las de los conductos estriados mayores), en este grupo. La cepa RAL del T. cruzi causó un cuadro general de atrofia general en la glándula sublingual, con numerosos nidos de parásitos.

Histometry of the sublingual gland in male and female mice (Mus musculus) infected with the RAL strain of the Chagas parasite, Trypanosoma cruzi. The aim of this work was to analyze histologically and histometrically the sublingual gland of mice infected with the RAL strain of T. cruzi, according to the sex. Swiss mice (Mus musculus) were inoculated with 2x104 blood trypomastigotes of the RAL strain of T. cruzi. in the peak of the parasitemia (12th day) the mice were sacrificed, and the sublingual glands were fixed in ALFAC. HE-stained histological sections were evaluated histometrically. The parasitemia was higher in females. Histopatologically, acini of the infected animals were smaller, with scanty production of secretion, and smaller striated ducts. The nuclei of the demilunes were smaller and showed amastigote nests in the cytoplasm. Karyometrically, nuclei of the acini, demilunes and striated ducts were smaller in the infected mice. Stereologically, it was observed that relative volumes of acini and ducts were smaller and, inversely, relative volumen were greater for the conjunctive tissue in the infected males. The surface densities of acini and ducts were bigger and the diameter and thickness of the wall were smaller in this group. On the other hand, relative volume of acini was smaller and those of the ducts and conjunctive tissue were bigger in the infected females. The diameter and thickness of the wall of acini were smaller, and those of the striated ducts were bigger in this group. The RAL strain of T. cruzi caused general atrophy in the sublingual gland, with numerous nests of parasites in the glandular parenchyma. Rev. Biol. Trop. 56 (2): 459-471. Epub 2008 June 30.

Mioplastias da musculatura extra-ocular podem ensejar aderências entre o músculo operado e os tecidos adjacentes, produzindo, não raro, estrabismos cicatriciais. Com intuito de se minimizar a ocorrência de aderências, investigaram-se os efeitos da mitomicina C, como agente antifibrótico, em concentrações ascendentes de 0,008, 0,02 e 0,04%, aplicada no per-operatório de mioplastias do reto superior do bulbo do olho de coelhos. Operaram-se 56 animais, que compuseram cinco grupos. No pós-operatório, instituíram-se limpeza com solução fisiológica das áreas operadas e profilaxia antimicrobiana e antiinflamatória, na forma de colírio. Avaliações clínica, histológica e imunoistoquímica, em que se estudou o fator de crescimento fibroblástico-básico (FGF-2), e estatística compuseram os métodos de análise dos resultados. Encontraram-se, clinicamente, mais aderências nos olhos-controle, comparativamente aos tratados, embora sem diferença estatística (P>0,05). À histologia, verificou-se que a mitomicina C ensejou retardo da cicatrização junto às áreas das mioplastias, notadamente no grupo que a recebeu, à concentração de 0,02%. A imunoistoquímica revelou marcação do FGF-2 em fibroblastos e macrófagos indistintamente entre os grupos. Com base nos resultados, permite-se admitir que a mitomicina C, nas concentrações em que foi empregada, retardou a cicatrização e, por conseguinte, o estrabismo secundário, sem ensejar efeitos colaterais.

Myoplasties of the extraocular muscle may cause adhesions between the operated muscle and the adjacent tissues, commonly generating cicatricial strabismus. With the purpose of reducing to a minimum the occurrence of adhesion, the effects of mitomycin C, an antifibrotic agent, were studied in concentrations of 0.008%, 0.02%, and 0.04% applied during intraoperative of myoplasties of the superior rectus muscle of rabbits. Fifty six animals were divided in five groups. During the postoperative, the operated areas were washed with physiological solution. Eye drop instillation to prevent inflammation and bacterial infection were used. The method to analyze the results consisted of clinical and histological evaluation and statistical analyzes. We also evaluated at the same time the amount of basic fibroblast growth factor (FGF-2) by immunohistochemical study. Clinically, more adhesions were found in the eyes of the control group than in the groups of treated eyes. However there was no significant statistics difference between the two groups (P>0.05). Histologically, mitomycin C caused a delayed cicatrization in the mioplastic areas, specially in the group who received the 0.02% concentration. The immunohistochemical showed FGF-2 marking in fibroblasts and macrophages, but between the groups there wasn't no difference. Based on those results, mitomycin C in the utilized concentrations was capable of delaying the cicatrization and consequently avoid the secondary strabismus without undesirable side effects.

The phytochemical investigation of trypanocidal extracts from leaves and stems of Conchocarpus heterophyllus (A. St.-Hil.) Kallunki & Pirani (Rutaceae) afforded new pyranoflavones along with the known compounds flavone, 7-methoxyflavone, 5-hydroxyflavone, haplotusine, 1-methyl-2-phenyl-4-quinolone alkaloid, beta-sitosterol, stigmasterol, and beta-sitosteryl benzoate. Their structures were established based on their spectral data. NMR data for the alkaloid haplotusine and the new pyranoflavones are described for the first time herein. These compounds were assayed on the tripomastigote forms of Trypanosoma cruzi. Among them, haplotusine and 1-methyl-2-phenyl-4-quinolone showed moderate values of IC50 136.9 and 144.9 mM, respectively.