IL7RA rs6897932 Polymorphism is Associated with Better CD4+ T-Cell Recovery in HIV Infected Patients Starting Combination Antiretroviral Therapy.

Interleukin-7 receptor subunit alpha (IL7RA) rs6897932 polymorphism is related to CD4+ recovery after combination antiretroviral therapy (cART), but no studies so far have analyzed its potential impact in patients with very low CD4+ T-cells count. We aimed to analyze the association between IL7RA rs6897932 polymorphism and CD4+ T-cells count restoration in HIV-infected patients starting combination antiretroviral therapy (cART) with CD4+ T-cells count <200 cells/mm3. We performed a retrospective study in 411 patients followed for 24 months with a DNA sample available for genotyping. The change in CD4+ T-cells count during the follow-up was considered as the primary outcome. The rs6897932 polymorphism had a minimum allele frequency (MAF) >20% and was in Hardy-Weinberg equilibrium (p = 0.550). Of 411 patients, 256 carried the CC genotype, while 155 had the CT/TT genotype. The CT/TT genotype was associated with a higher slope of CD4+ T-cells recovery (arithmetic mean ratio; AMR = 1.16; p = 0.016), higher CD4+ T-cells increase (AMR = 1.19; p = 0.004), and higher CD4+ T-cells count at the end of follow-up (AMR = 1.13; p = 0.006). Besides, rs6897932 CT/TT was related to a higher odds of having a value of CD4+ T-cells at the end of follow-up ≥500 CD4+ cells/mm3 (OR = 2.44; p = 0.006). After multiple testing correction (Benjamini-Hochberg), only the increase of ≥ 400 CD4+ cells/mm3 lost statistical significance (p = 0.052). IL7RA rs6897932 CT/TT genotype was related to a better CD4+ T-cells recovery and it could be used to improve the management of HIV-infected patients starting cART with CD4+ T-cells count <200 cells/mm3.

Bacterial translocation and clinical progression of HCV-related cirrhosis in HIV-infected patients.

The aim of the study was to evaluate whether bacterial translocation (BT) predicts the clinical outcome in HIV/HCV-coinfected patients with compensated cirrhosis. A cohort of 282 HIV/HCV-coinfected patients with cirrhosis and no previous liver decompensation (LD) was recruited. Serum levels of the DNA sequences encoding the well-conserved 16S rRNA subunit (16S rDNA), the lipopolysaccharide (LPS) and soluble CD14 (sCD14) at diagnosis of cirrhosis were measured. Primary endpoint was the emergence of the first LD and/or death of any cause. Secondary endpoints were LD, liver-related death (LRD) and death of any cause. After a median (Q1-Q3) follow-up of 51 (27-72) months, 67 patients (24%; 95% CI: 19-29) developed their first LD or died during follow-up. Baseline levels of 16S rDNA, LPS and sCD14 were not associated with the probability of developing the primary endpoint of the study. The mean (SD) survival time free of LD and/or death according to levels of 16S rDNA (<83, 83-196, 197-355, >355 [copies/µL]) was 78 (5), 72 (5), 81 (4) and 82 (4) months, respectively (P = .5). The corresponding figures for LPS (<0.1, 0.1-0.6, 0.6-1.5, > 1.5 [IU/mL]) were 76 (5), 71 (5), 77 (5) and 81 (4) months, respectively (P = .4). Baseline levels of BT serum markers were not associated with any of the secondary endpoints analysed in the study. Thus, BT does not seem to be a relevant predictor of clinical outcome in HIV/HCV-coinfected patients with compensated cirrhosis.

Avaliação da atividade angiogênica da solução aquosa do barbatimão (Stryphnodendron adstringens) / Angiogenic activity of the aqueous solution of Barbatimão (Stryphnodendron adstringens)

RESUMO O Barbatimão (Stryphnodendron adstringens) planta medicinal encontrada no bioma Cerrado apresenta propriedades físico-químicas que lhe garante importantes atividades farmacológicas tais como: anti-inflamatória, analgésica e uma atividade protetora da mucosa gástrica. A casca do tronco é a principal matéria-prima usada para o desenvolvimento de produtos medicinais. Neste estudo, o objetivo foi investigar a influência da solução aquosa da casca do barbatimão no processo de formação de vasos sanguíneos na membrana corioalontoide de ovo embrionado de galinha. Foram utilizadas 30g da casca triturada em um litro de água. Este processo permitiu a obtenção da Solução Aquosa de Barbatimão - SAB em uma concentração de 30mg/mL. A atividade angiogênica da solução aquosa do barbatimão foi avaliada mediante realização de testes laboratoriais “in vivo”, utilizando como modelo experimental a membrana do ovo embrionado de galinha (MCA). Utilizou-se como controle indutor o Regederm®, o qual apresenta atividade angiogênica conhecida. Os resultados demonstraram que a SAB apresentou um percentual de vascularização na MCA de (50.4%) não tendo diferença (p>0,05) aos valores detectados no controle indutor (52,9%). Com os resultados obtidos, percebe-se que o barbatimão apresenta atividade angiogênica no modelo experimental utilizado.

ABSTRACT The Barbatimão (Stryphnodendron adstringens) medicinal plant found in the Cerrado biome has physicochemical properties which guarantee important pharmacological activities such as anti-inflammatic, analgesic and protective activities of gastric mucosa. The bark of the trunk is the main raw material used for the development of medicinal products. In this study, the objective was to investigate the influence of the aqueous solution of barbatimão bark in the formation of blood vessels in the membrane of embryonated chicken egg corioalontoid. 30g of shredded bark was used in one liter of water. This process enabled the obtention of aqueous Barbatimão - BSA at a concentration of 30mg / ml. The angiogenic activity of the aqueous solution of barbatimão was assessed by laboratory testing “in vivo”, using the chorioallantoic membrane of embryonated chicken egg (MCA) as an experimental model. TheRegederm® controlinductor was used, which exhibits known angiogenic activity. The results showed that the percentage of BSA showed a vascularization of the MCA (50.4%) there was no difference (p> 0.05) in the values detected in the control inductor (52.9%). With the obtained results, it is clear that barbatimão shows angiogenic activity under the experimental model used.

Association between IL7RA polymorphisms and the successful therapy against HCV in HIV/HCV-coinfected patients.

Interleukin-7 (IL-7) is a critical factor in maintaining or inducing effective antiviral CD4+ and CD8+ T-cell responses. The aim of this study was to examine the association of interleukin-7 receptor-α (IL7RA) polymorphisms with a sustained virologic response (SVR) after hepatitis C virus (HCV) therapy with pegylated interferon-alpha plus ribavirin (pegIFNα/ribavirin) in 177 human immunodeficiency virus (HIV)/HCV-coinfected patients. We performed a retrospective study in 177 naïve patients who started HCV treatment. The IL7RA rs6897932, rs987106, and rs3194051 polymorphisms were genotyped by the GoldenGate® assay. An SVR was defined as undetectable HCV viral load through 24 weeks after the end of HCV treatment. The highest SVR rate was found in patients with the rs6897932 CC (p = 0.029) and rs3194051 GG (p = 0.002) genotypes, and HCV genotypes 2/3 (GT2/3) infected patients with the rs987106 AA genotype (p = 0.048). Additionally, carriers of the rs3194051 GG genotype had a higher likelihood of achieving an SVR [adjusted odds ratio (aOR) = 5.32; 95 % confidence interval (CI) = 1.07-26.94; p = 0.040] than patients with the rs3194051 AA/AG genotype, while rs6897932 CC (aOR = 0.63; p = 0.205) and rs987106 AA (aOR = 0.60; p = 0.213) were not significant. Moreover, three major haplotypes were found: 46.6 % for CTA, 32.4 % for CAG, and 20.7 % for TAA haplotypes. Patients infected with GT2/3 and carriers of the CTA haplotype had lower odds of achieving an SVR (aOR = 0.08; p = 0.004) and the CAG haplotype (favorable alleles) had higher odds of achieving an SVR than other haplotypes (aOR = 21.96; p < 0.001). IL7RA polymorphisms seem to play a significant role in the virological response to pegIFNα/ribavirin therapy in HIV/HCV-coinfected patients, in particular among patients infected with HCV GT2/3.

Genetic diversity and population structure of different varieties of Morada Nova hair sheep from Brazil.

The aim of this study was to analyze genetic diversity and population structure among varieties of White (N = 40), Red (N = 32), and Black (N = 31) Morada Nova hair sheep from flocks in the northeastern Brazilian semiarid region. Fifteen nuclear microsatellite markers and two regions of mitochondrial DNA were used. The intra-population analysis demonstrated that the White variety had higher diversity, while the Red variety had the lowest values. The Bayesian analysis to assess the genetic population structure allowed differentiation between White, Red, and Black varieties, and revealed a tendency towards sub-structuring in the White variety flocks from the States of Ceará and Paraíba. The results of analyses of molecular variance showed that the greatest genetic structure was found when comparing flocks rather than varieties (8.59 vs 6.64% of the total variation, P < 0.001). Based on genetic distance, Dtl, both the dendrogram analysis and the principal coordinate analysis showed the formation of two main groups: one composed of White and another of Black and Red individuals. Five and two haplotypes were found for the D-loop region and the ND5 gene, respectively. A haplotype unique to the Red variety was found in the D-loop region and a variety haplotype unique to the Black variety was found in the ND5 gene; however, these frequencies were low and therefore require further validation. These results support the existence of substantial differences between the Red and White varieties and should be used as separate genetic resources and to improve conservation programs.

European mitochondrial haplogroups are not associated with hepatitis C virus (HCV) treatment response in HIV/HCV-coinfected patients.

OBJECTIVES: Mitochondria are multifunctional organelles with a key role in the innate immune response against viral infections. Mitochondrial DNA (mtDNA) haplogroups have been related to AIDS progression and CD4 T-cell recovery in HIV-infected patients, and to a delay in the development of liver fibrosis in HIV/hepatitis C virus (HCV)-coinfected patients. We performed a study to investigate whether mtDNA haplogroups may be associated with HCV treatment response in HIV/HCV-coinfected patients on pegylated interferon (pegIFN) plus ribavirin (RBV). METHODS: We performed a retrospective study in 304 patients who completed a course of HCV therapy. mtDNA polymorphisms were genotyped using Sequenom's MassARRAY platform. The interleukin-28B (IL-28B) polymorphism (rs12980275) was genotyped using the GoldenGate® assay. Sustained virological response (SVR) was defined as an undetectable HCV viral load at week 24 after the end of treatment. The statistical analysis was carried out using on-treatment data. RESULTS: The SVR rates were 52.6% (160 of 304) for all patients, and 37.8% (46 of 201) for patients with HCV genotype 1 or 4 vs. 81.4% (83 of 102) for patients with HCV genotype 2 or 3 (P < 0.001). No significant associations were found between mtDNA haplogroup and SVR when all patients were included in the analysis and when patients were stratified by HCV genotype (i.e. those with genotypes 1/4 and 2/3 analysed separately) or IL-28B rs12980275 genotype. CONCLUSIONS: European mtDNA haplogroups were not related to HCV treatment response in HIV/HCV-coinfected patients on pegIFN-α/RBV therapy.

[Validity and reliability of the spanish EQ-5D-Y proxy version]. / Validez y fiabilidad de la versión proxy del EQ-5D-Y en español.

INTRODUCTION: A proxy version of the EQ-5D-Y, a questionnaire to evaluate the Health Related Quality of Life (HRQoL) in children and adolescents, has recently been developed. There are currently no data on the validity and reliability of this tool. The objective of this study was to analyze the validity and reliability of the EQ-5D-Y proxy version. METHODOLOGY: A core set of self-report tools, including the Spanish version of the EQ-5D-Y were administered to a group of Spanish children and adolescents drawn from the general population. A similar core set of internationally standardized proxy tools, including the EQ-5D-Y proxy version were administered to their parents. Test-retest reliability was determined, and correlations with other generic measurements of HRQoL were calculated. Additionally, known group validity was examined by comparing groups with a priori expected differences in HRQoL. The agreement between the self-report and proxy version responses was also calculated. RESULTS: A total of 477 children and adolescents and their parents participated in the study. One week later, 158 participants completed the EQ-5D-Y/EQ-5D-Y proxy to facilitate reliability analysis. Agreement between the test-retest scores was higher than 88% for EQ-5D-Y self-report, and proxy version. Correlations with other health measurements showed similar convergent validity to that observed in the international EQ-5D-Y. Agreement between the self-report and proxy versions ranged from 72.9% to 97.1%. CONCLUSIONS: The results provide preliminary evidence of the reliability and validity of the EQ-5D-Y proxy version.

IL28RA polymorphism (rs10903035) is associated with insulin resistance in HIV/HCV-coinfected patients.

Hepatitis C virus (HCV) infection is associated with insulin resistance (IR), although mechanisms leading to IR in these patients are not completely understood. The aim of this study was to evaluate the association of interleukin 28B (IL28B) and interleukin 28 receptor alpha (IL28RA) polymorphisms with IR among human immunodeficiency virus (HIV)/HCV-coinfected patients. We carried out a cross-sectional study on 203 patients. IL28B (rs8099917) and IL28RA (rs10903035) polymorphisms were genotyped by GoldenGate(®) assay. IR was defined as homeostatic model assessment (HOMA) values ≥3.00. Univariate and multivariate generalized linear models (GLM) were used to compare HOMA values and the percentage of patients with IR according to IL28B and IL28RA genotypes. In total, 32% (n = 65/203) of the patients had IR. IL28B rs8099917 TT was not significantly associated with HOMA values and IR. In contrast, rs10903035 AA was significantly associated with high HOMA values taking into account all patients (P = 0.024), as well as the subgroups of patients with significant fibrosis (P = 0.047) and infected with HCV genotype 3 (P = 0.024). Additionally, rs10903035 AA was significantly associated with IR (HOMA ≥3.00) in all patients (adjusted odds ratio (aOR) = 2.02; P = 0.034), in patients with significant fibrosis (aOR = 2.86; P = 0.039) and HCV genotype 3 patients (aOR = 4.89; P = 0.031). In conclusions, IL28RA polymorphism (rs10903035) seems to be implicated in the glucose homeostasis because AA genotype increases the likelihood of IR, but this association was different depending on hepatic fibrosis and HCV genotype.

Lorazepam photofate under photolysis and TiO2-assisted photocatalysis: identification and evolution profiles of by-products formed during phototreatment of a WWTP effluent.

This manuscript reports on the study of Lorazepam (LZP) phototransformation pathways under artificial UV and natural solar irradiation, through photolytic and TiO2-assisted photocatalytic processes. Three experimental set-ups were employed: two lab-scale photoreactors, each provided with an UV lamp (one medium pressure mercury lamp and one blacklight blue lamp), and a pilot-scale Solar Plant with Compound Parabolic Collectors (CPCs). Samples collected along the different phototreatment experiments were analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UHPLC/QqToF-MS). The key assumption of the analytical approach was that related compounds (LZP and its by-products (LBPs)) provide identical "diagnostic fragment ions". Identification was also based on the chlorine atoms specific isotopic pattern, as well as accurate masses. Six major LBPs were identified and elucidated, with nominal [M + H](+) masses of 337, 303, 319, 275, 291 and 293 Da. The proposed LZP photodegradation mechanism included the initial opening of the diazepinone seven-membered ring, followed by a rearrangement into a highly stabilized six-membered aromatic ring and subsequent cleavage and/or hydroxylation reactions. The evolution profiles of LBPs were described for each of the three experimental prototypes and the CPCs Solar Pilot Plant proved to be the most efficient one. Finally, LZP photocatalytic degradation was further assessed on a municipal effluent, where the photoproducts generated showed to be more persistent than LZP itself.

Multistage treatment system for raw leachate from sanitary landfill combining biological nitrification-denitrification/solar photo-Fenton/biological processes, at a scale close to industrial--biodegradability enhancement and evolution profile of trace pollutants.

A multistage treatment system, at a scale close to the industrial, was designed for the treatment of a mature raw landfill leachate, including: a) an activated sludge biological oxidation (ASBO), under aerobic and anoxic conditions; b) a solar photo-Fenton process, enhancing the bio-treated leachate biodegradability, with and without sludge removal after acidification; and c) a final polishing step, with further ASBO. The raw leachate was characterized by a high concentration of humic substances (HS) (1211 mg CHS/L), representing 39% of the dissolved organic carbon (DOC) content, and a high nitrogen content, mainly in the form of ammonium nitrogen (>3.8 g NH4(+)-N/L). In the first biological oxidation step, a 95% removal of total nitrogen and a 39% mineralization in terms of DOC were achieved, remaining only the recalcitrant fraction, mainly attributed to HS (57% of DOC). Under aerobic conditions, the highest nitrification rate obtained was 8.2 mg NH4(+)-N/h/g of volatile suspended solids (VSS), and under anoxic conditions, the maximum denitrification rate obtained was 5.8 mg (NO2(-)-N + NO3(-)-N)/h/g VSS, with a C/N consumption ratio of 2.4 mg CH3OH/mg (NO2(-)-N + NO3(-)-N). The precipitation of humic acids (37% of HS) after acidification of the bio-treated leachate corresponds to a 96% DOC abatement. The amount of UV energy and H2O2 consumption during the photo-Fenton reaction was 30% higher in the experiment without sludge removal and, consequently, the reaction velocity was 30% lower. The phototreatment process led to the depletion of HS >80%, of low-molecular-weight carboxylate anions >70% and other organic micropollutants, thus resulting in a total biodegradability increase of >70%. The second biological oxidation allowed to obtain a final treated leachate in compliance with legal discharge limits regarding water bodies (with the exception of sulfate ions), considering the experiment without sludge. Finally, the high efficiency of the overall treatment process was further reinforced by the total removal percentages attained for the identified organic trace contaminants (>90%).

IL28RA polymorphism is associated with early hepatitis C virus (HCV) treatment failure in human immunodeficiency virus-/HCV-coinfected patients.

Due to the poor rate of response to hepatitis C virus (HCV) with pegylated interferon and ribavirin treatment in HCV/HIV coinfected patients, key factors for predicting failure would be useful. We performed a retrospective study on 291 patients on HCV treatment, who had early virological response (EVR) data. IL28B and IL28RA polymorphisms were performed using the GoldenGate(®) assay. Unfavourable genotypes at IL28B (rs12980275 AG/GG and rs8099917 GT/GG) and an unfavourable allele at IL28RA (rs10903035 G) were associated with early treatment failure. However, only the rs12980275 AG/GG genotype and rs10903035 G allele remained independently associated with early failure in the overall population (OR = 4.15 (95% CI = 1.64-10.54) and OR = 2.00 (95% CI = 1.19-3.36), respectively) as well as in GT1/4 patients (OR = 5.07 (95% CI = 1.81-14.22) and OR = 2.03 (95% CI = 1.13-3.66), respectively). Next, a decision tree showed early treatment failure increased from 37.1% to 65.5% when the unfavourable rs12980275 AG/GG and rs10903035 AG/GG genotypes and HCV-RNA≥ 500.000 IU/mL were taken into account in GT1/4 patients. In contrast, the failure rate decreased from 37.1% to 11.9% when the favourable rs12980275 AA and rs10903035 AA genotypes were detected. The percentage of patients correctly classified was 78.4%, and AUROC was 0.802 ± 0.028. Regarding GT3 patients, the presence of the GCGCA haplotype (all unfavourable alleles) was associated with early treatment failure, while no association was observed for the IL28B polymorphisms. In conclusion, the IL28RA polymorphism was associated with early treatment failure independently of the IL28B SNPs. The combination of IL28B and IL28RA polymorphisms might be a valuable tool for predicting early treatment failure before starting HCV treatment.

[Translation and cultural adaptation to spanish of the questionnaire EQ-5D-Y Proxy version]. / Traducción y adaptación cultural al español de la versión Proxy del cuestionario EQ-5D-Y.

INTRODUCTION: The Health Related Quality of Life (HRQoL) questionnaire EQ-5D-Y for children and adolescents has been development and validated recently for its use in Spanish. However, the Spanish Proxy version of this tool has not yet been developed. The aim of this study was to translate and culturally adapt the international version of EQ-5D-Y Proxy into Spanish to measure the health related quality of life in the child and adolescent population. MATERIAL AND METHOD: This study has been developed in the following phases: a) Transcultural adaptation of the international questionnaire by means of a direct translation to Spanish and back-translation to English, b) evaluation of the clarity, acceptability and familiarity of the first version of the questionnaire using probing and paraphrasing methods in 30 parents of children and adolescents distributed by sex and age. MAIN RESULTS: The Spanish EQ-5Y Proxy version was obtained. The interviewed participants indicated an excellent comprehensibility of the items and the perceived difficulty was less than 2 in all dimensions of the questionnaire, except in mobility where it was a slightly higher. CONCLUSIONS: The Spanish EQ-5D-Y Proxy version has shown to be well understandable and adapted to Spanish parents of children and adolescents. Its easy administration makes this questionnaire potentially useful in different fields.

Biodegradability enhancement of a pesticide-containing bio-treated wastewater using a solar photo-Fenton treatment step followed by a biological oxidation process.

This work proposes an efficient combined treatment for the decontamination of a pesticide-containing wastewater resulting from phytopharmaceutical plastic containers washing, presenting a moderate organic load (COD=1662-1960 mg O2 L⁻¹; DOC=513-696 mg C L⁻¹), with a high biodegradable organic carbon fraction (81%; BOD5=1350-1600 mg O2 L⁻¹) and a remaining recalcitrant organic carbon mainly due to pesticides. Nineteen pesticides were quantified by LC-MS/MS at concentrations between 0.02 and 45 mg L⁻¹ (14-19% of DOC). The decontamination strategy involved a sequential three-step treatment: (a) biological oxidation process, leading to almost complete removal of the biodegradable organic carbon fraction; (b) solar photo-Fenton process using CPCs, enhancing the bio-treated wastewater biodegradability, mainly due to pesticides degradation into low-molecular-weight carboxylate anions; (c) and a final polishing step to remove the residual biodegradable organic carbon, using a biological oxidation process. Treatment performance was evaluated in terms of mineralization degree (DOC), pesticides content (LC-MS/MS), inorganic ions and low-molecular-weight carboxylate anions (IC) concentrations. The estimated phototreatment energy necessary to reach a biodegradable wastewater, considering pesticides and low-molecular-weight carboxylate anions concentrations, Zahn-Wellens test and BOD5/COD ratio, was only 2.3 kJ(UV) L⁻¹ (45 min of photo-Fenton at a constant solar UV power of 30 W m⁻²), consuming 16 mM of H2O2, which pointed to 52% mineralization and an abatement higher than 86% for 18 pesticides. The biological oxidation/solar photo-Fenton/biological oxidation treatment system achieved pesticide removals below the respective detection limits and 79% mineralization, leading to a COD value lower than 150 mg O2 L⁻¹, which is in agreement with Portuguese discharge limits regarding water bodies.

Cleanup strategies and advantages in the determination of several therapeutic classes of pharmaceuticals in wastewater samples by SPE-LC-MS/MS.

This work describes the development and validation of an offline solid-phase extraction with simultaneous cleanup capability, followed by liquid chromatography-(electrospray ionisation)-ion trap mass spectrometry, enabling the concurrent determination of 23 pharmaceuticals of diverse chemical nature, among the most consumed in Portugal, in wastewater samples. Several cleanup strategies, exploiting the physical and chemical properties of the analytes vs. interferences, alongside with the use of internal standards, were assayed in order to minimise the influence of matrix components in the ionisation efficiency of target analytes. After testing all combinations of adsorbents (normal-phase, ion exchange and mixed composition) and elution solvents, the best results were achieved with the mixed-anion exchange Oasis MAX cartridges. They provided recovery rates generally higher than 60%. The precision of the method ranged from 2% to 18% and 4% to 19% (except for diclofenac (22%) and simvastatin (26%)) for intra- and inter-day analysis, respectively. Method detection limits varied between 1 and 20 ng L(-1), while method quantification limits were <85 ng L(-1) (both excluding ibuprofen). This analytical method was applied to gather preliminary results on influents and effluents of two wastewater treatment plants (WWTPs) located in the urban region of Porto (Portugal). Typically, paracetamol, hydrochlorothiazide, furosemide, naproxen, ibuprofen, diclofenac and bezafibrate were detected in concentrations ranging from 1 to 20 µg L(-1), while gemfibrozil, simvastatin, ketoprofen, azithromycin, bisoprolol, lorazepam and paroxetine were quantified in levels below 1 µg L(-1). These WWTPs were given particular attention since they discharge their effluents into the Douro river, where water is extracted for the production of drinking water. Some sampling spots in this river were also analysed.

Trypanosoma caninum n. sp. (Protozoa: Kinetoplastida) isolated from intact skin of a domestic dog ( Canis familiaris) captured in Rio de Janeiro, Brazil.

An unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15.2%) and DMEM (9.2%) media containing 5% FCS, with a mean length of these forms of 43.0 and 36.0 mum, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24salpha rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.

Endothelium-dependent vasodilator effect of Euterpe oleracea Mart. (Açaí) extracts in mesenteric vascular bed of the rat.

Açai (Euterpe oleracea Mart.) a fruit from the Amazon region, largely consumed in Brazil is rich in polyphenols. Experiments were undertaken to determine whether hydro-alcoholic extract obtained from stone of açaí induces a vasodilator effect in the rat mesenteric vascular bed precontracted with norepinephrine (NE) and, if so, to elucidate the underlying mechanism. Açai stone extract (ASE, 0.3-100 microg) induced a long-lasting endothelium-dependent vasodilation that was significantly reduced by N(G)-nitro-l-arginine methyl ester (l-NAME) and (1)H-[1,2,3] oxadiazolo [4,4-a] quinoxalin-l-one (ODQ) and abolished by KCl (45 mM) plus l-NAME. In vessels precontrated with NE and KCl (45 mM) or treated with K(Ca)(+2) channel blockers (charybdotoxin plus apamin), the effect of ASE was significantly reduced. However this effect is not affect by indomethacin, glybenclamide and 4-aminopiridine. Atropine, pyrilamine, yohimbine and HOE 140 significantly reduced the vasodilator effect of acetylcholine, histamine, clonidine and bradykinin, respectively, but did not change the vasodilator effect of ASE. In cultured endothelial cells ASE (100 microg/mL) induced the formation of NO that was reduced by N(G)-nitro-l-arginine (l-NA, 100 microM). The present study demonstrates that the vasodilator effect of ASE is dependent on activation of NO-cGMP pathway and may also involve endothelium-derived hyperpolarizing factor (EDHF) release. The vasodilator effect suggest a possibility to use ASE as a medicinal plant, in the treatment of cardiovascular diseases.

Molecular karyotype and chromosomal localization of genes encoding beta-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli.

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of beta-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of beta-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.