Specific activation of CD4- CD8- double-negative T cells by Trypanosoma cruzi-derived glycolipids induces a proinflammatory profile associated with cardiomyopathy in Chagas patients.

Cardiomyopathy is the most severe outcome of Chagas disease, causing more than 12 000 deaths/year. Immune cells participate in cardiomyopathy development either by direct tissue destruction, or by driving inflammation. We have shown that CD4- CD8- [double-negative (DN)] T cells are major sources of inflammatory and anti-inflammatory cytokines, associated with the cardiac (CARD) and indeterminate (IND) forms of Chagas disease, respectively. Here, we sought to identify Trypanosoma cruzi-derived components that lead to activation of DN T cells in Chagas patients. Glycolipid (GCL), lipid (LIP) and protein-enriched (PRO) fractions derived from trypomastigote forms of T. cruzi were utilized to stimulate cells from IND and CARD patients to determine DN T cell activation by evaluating CD69 and cytokine expression. We observed that GCL, but not LIP or PRO fractions, induced higher activation of DN T cells, especially T cell receptor (TCR)-γδ DN T, from IND and CARD. GCL led to an increase in tumour necrosis factor (TNF) and interleukin (IL)-10 expression by TCR-γδ DN T cells from IND, while inducing IFN-γ expression by TCR-γδ DN T cells from CARD. This led to an increase in the ratio IFN-γ/IL-10 in TCR-γδ DN T cells from CARD, favouring an inflammatory profile. These results identify GCL as the major T. cruzi component responsible for activation of DN T cells in chronic Chagas disease, associated predominantly with an inflammatory profile in CARD, but not IND. These findings may have implications for designing new strategies of control or prevention of Chagas disease cardiomyopathy by modulating the response to GCL.

Plasma biomarkers profile of female dogs with mammary carcinoma and its association with clinical and pathological features.

The immunological biomarkers profiles were evaluated using Luminex as putative measures to monitor canine mammary carcinomas (MCs). Forty female dogs were categorized into benign mixed tumour (MC-BMT = 28) and mammary carcinoma (MC=12). The ascendant biomarker signatures were used to compare the groups. For example, a higher frequency of MC-BMT animals producing IL-6, CXCL-8 and CXCL-10 was observed, whereas for the MC group IL-2 and CXCL-8 were detected. MC-BMT animals without metastasis had an increase in the levels of IL-2, CXCL-8, CXCL-10, IL-6, TNF-α, IL-15 and a decrease in IL-10 and CXCL-8. MC-BMT animals with metastasis showed only an increase in CXCL-10 and a decrease in IL-18. After comparing the ascendant signatures following the presence of metastasis in both groups, a higher frequency of dogs exhibiting IL-10 production was observed. Pearson correlation (P = 0.0273) and receiver operating characteristic (ROC) curve analysis revealed that this pattern was associated with worse outcome and lower survival rates in MC animals.

Avaliação da toxicidade aguda do extrato das cascas de Pithecellobium cochliocarpum (Gomez) Macbr / Evaluation of acute toxicity of the Pithecellobium cochliocarpum (Gomez) Macbr bark extract

O uso popular, e mesmo o tradicional, não são suficientes para validar as plantas medicinais como medicamentos eficazes e seguros. Para melhor entendimento, é necessário avaliar a relação risco/benefício de seu uso, por meio de estudos toxicológicos. O objetivo desta pesquisa foi estimar a toxicidade aguda do extrato etanólico das cascas secas de Pithecellobium cochliocarpum (Gomez) Macbr através da obtenção da dose letal (DL50) em roedores, e da Concentração letal (CL50) frente à Artemia salina Leach. Foram realizados experimentos por via oral e intraperitoneal utilizando camundongos fêmeas albinos Swiss (Mus musculus) (n=6). Por via oral foram administradas 3 doses (1.000, 3.000 e 5.000 mg Kg-1) e por via entraperitoneal, 5 doses (155, 160, 176, 345,6 e 414,72 Kg-1). Os sinais comportamentais foram avaliados durante uma hora após a administração do extrato, ficando em observação até 48 horas. O número de óbitos foi quantificado para posterior cálculo da DL50. A administração por via intraperitoneal foi realizada em intervalo de 5 minutos para cada animal. Nos ensaios de toxicidade por via oral a solução foi introduzida por via intragástrica através de cânula metálica acoplada a seringa (gavagem) no mesmo intervalo de tempo utilizado pela via intraperitoneal. Os animais do grupo de administração oral apresentaram algumas reações, porém não letais até a dose de 5.000 mg Kg-1. A DL50 para a via intraperitoneal foi 257, 49 mg Kg-1 (muito tóxico, grau 4) (Schuartsman, 1980). A CL50 (543,5 µg Kg-1) demonstrou ser tóxica frente à A. salina. Conclui-se que sob condições agudas de exposição, o extrato do Pithecellobium cochliocarpum é um agente tóxico, devendo ser considerado como tal, dependendo da dose administrada ou absorvida, do etempo e frequência de exposição e das vias de administração.

The popular use, and even the traditional one, is not enough to validate medicinal plants as effective and safe medicines. For a better understanding, it is necessary to assess the risk / benefit ratio of their use through toxicological studies. The aim of this work was to evaluate the acute toxicity of Pithecellobium cochliocarpum (Gomez) Macbr dried bark ethanolic extract through its lethal dose (LD50), in mice, and lethal concentration (LC50) in relation to Artemia salina Leach. Experiments were performed by oral and intraperitoneal route using female Swiss albino mice (Mus musculus) (n = 6). The first three doses were given orally (1,000, 3,000 and 5,000 mg kg-1) and the last five doses were given intraperitoneally (155, 160, 176, 345.6 and 414.72 Kg-1). The behavioral signs were evaluated one hour after administration of the extract, being observed up to 48 hours. The number of deaths was quantified for subsequent calculation of LD50. The intraperitoneal administration was carried out at an interval of 5 minutes for each animal. For the oral toxicity test, the solution was introduced in the digestive system of the animals through a metal cannula coupled to a syringe (gavage) at the same time interval used for the intraperitoneal route. The animals from the oral group presented some reactions, but they were not lethal up to the dose of 5.000 mg kg-1. The LD50 for the intraperitoneal group was 257.49 mg kg-1 (very toxic, grade 4) (Schuartsman, 1980). The LC50 (543.5 mg kg-1) was toxic to A. salina. We can conclude that, under acute conditions of exposure, the Pithecellobium cochliocarpum extract is a poisonous agent and should be considered as such depending on the administered or absorbed dose, the time and frequency of exposure, and the administration routes.

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease.

Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

PP147. Prevalence of hypertensive syndromes according to pregnancy age.

INTRODUCTION: The decision of a woman to get pregnant at later age of her reproductive cycle has be a phenomenon around the world. Epidemiology data show frequent increase of clinical complications in direct proportion in advanced age motherhood, hypertensive disturbances being more prevalent. OBJECTIVES: Analyse the prevalence of hypertensive syndromes in pre-determined age groups at Hospital Guilherme Álvaro in Santos, São Paulo, Brazil showing the different segments in each one. METHODS: From data collected in the outpatient department of Hospital Guilherme Álvaro of High Risk Pre-natal between 04/06/2008 and 30/05/2011, a prevalence transversal study was carried out where data were obtained from 628 patients aged between 16 and 46years. Procedures of homogeny analysts were set out, always collecting data such as age and disorder for high risk gestation. According to age, patients were divided into groups: precocious (up to 19years old), middle age (between 20 and 34) and late pregnancies (over 35). RESULTS: In the precocious pregnancies, clinical illnesses/no hypertension (31%) were observed in first place, 25% (8) twin pregnancy in second place, 19% (6) showed hypertensive disturbances. Concerning pregnancies between 20 and 34years old, 36% (144 patients) showed hypertensive syndromes, 23% (92 patients) showed endocrine disturbances, 22% (90 patients) showed clinical illnesses/no hypertension, and 9% twin pregnancy. Regarding late pregnancies, the most frequent disturbance was isolated hypertensive syndromes: 44% (88 patients) in first place, only endocrine disturbances, 24% (47 patients) in second place followed by association between hypertensive syndromes and endocrinopathy with 13% (26 patients). CONCLUSION: About precocious pregnancies, greater prevalence showed clinical illnesses/no hypertension, whereas middle age and late pregnancies showed greater hypertensive syndrome prevalence, results, which are compatible with other studies, have been observed that due to advance of age, hypertensive syndromes are more frequent. For late pregnancies, the prevalence of clinical illnesses/no hypertension was a lower percentage regarding the other two groups: precocious pregnancies (31%=10 women), middle age pregnancies (22%=90 women), and late pregnancies (2%=7 women). Considering the fact that the occurrence of pregnancy is more and more late in life, it can be concluded that the professionals must be prepared to attend pregnancies on women with hypertensive disturbances and their eventual complications.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106 percent increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95 percentCI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease.

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106% increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95%CI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts.

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one ß-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.

Phlebotomine sand flies (Diptera: Psychodidae) and Leishmania infection in Gafanhoto Park, Divinópolis, Brazil.

The potential of Gafanhoto Park as an American cutaneous leishmaniasis (ACL) focus was evaluated by examination of sand fly vectors of the Leishmania parasite. This forest remnant is located in a periurban area of Divin6polis, Brazil, where autochthonous cases of ACL have been reported. Sand fly populations were monitored over a 2-yr period (2006-2008) by using light traps (HP and Shannon). During systematic collections with HP traps, 824 specimens in total (342 males and 482 females) of 21 species were captured. Most prevalent species were as follows: Brumptomyia brumpti (Larrouse), Lutzomyia aragaoi (Costa Lima), Lutzomyia lutziana (Costa Lima), Lutzomyia sordellii (Shannon & Del Ponte), and Lutzomyia whitmani (Antunes & Coutinho). Using Shannon traps, 257 specimens representing 15 species were collected (159 females and 98 males), with a high prevalence of L. whitmani and Lutzomyia neivai (Pinto), both vectors of Leishmania braziliensis (Vianna). To ascertain the level of natural infection, a sample of females captured in Shannon traps was assayed for the presence of Leishmania by using polymerase chain reaction-restriction fragment length polymorphism, where 39% of insects were positive. The most infected species was L. whitmani (29 sand flies; 18.2%), followed by L. neivai (21; 13.2%), Lutzomyia christenseni (Young & Duncan) (five; 3.1%), Lutzomyia pessoai (Coutinho & Barreto) (three; 1.9%), L. aragaoi (one; 0.6%), Lutzomyia fischeri (Pinto) (one; 0.6%), Lutzomyia lenti (Mangabeira) (one; 0.6%), L. lutziana (one; 0.6%), and Lutzomyia monticula (Costa Lima) (one; 0.6%). The finding of potential and incriminated vectors naturally infected with Leishmania reinforces the need of epidemiologic surveillance in the area.