In Vitro And In Vivo Anti-angiogenic Effects Of Hydroxyurea.

Hydroxyurea (HU), or hydroxycarbamide, is used for the treatment of some myeloproliferative and neoplastic diseases, and is currently the only drug approved by the FDA for use in sickle cell disease (SCD). Despite the relative success of HU therapy for SCD, a genetic disorder of the hemoglobin β chain that results in red-cell sickling, hemolysis, vascular inflammation and recurrent vasoocclusion, the exact mechanisms by which HU actuates remain unclear. We hypothesized that HU may modulate endothelial angiogenic processes, with important consequences for vascular inflammation. The effects of HU (50-200 μM; 17-24 h) on endothelial cell functions associated with key steps of angiogenesis were evaluated using human umbilical vein endothelial cell (HUVEC) cultures. Expression profiles of the HIF1A gene and the miRNAs 221 and 222, involved in endothelial function, were also determined in HUVECs following HU administration and the direct in vivo antiangiogenic effects of HU were assessed using a mouse Matrigel-plug neovascularization assay. Following incubation with HU, HUVECs exhibited high cell viability, but displayed a significant 75% inhibition in the rate of capillary-like-structure formation, and significant decreases in proliferative and invasive capacities. Furthermore, HU significantly decreased HIF1A expression, and induced the expression of miRNA 221, while downregulating miRNA 222. In vivo, HU reduced vascular endothelial growth factor (VEGF)-induced vascular development in Matrigel implants over 7 days. Findings indicate that HU is able to inhibit vessel assembly, a crucial angiogenic process, both in vitro and in vivo, and suggest that some of HU's therapeutic effects may occur through novel vascular mechanisms.

High-throughput Analysis By Sp-ldi-ms For Fast Identification Of Adulterations In Commercial Balsamic Vinegars.

Balsamic vinegar (BV) is a typical and valuable Italian product, worldwide appreciated thanks to its characteristic flavors and potential health benefits. Several studies have been conducted to assess physicochemical and microbial compositions of BV, as well as its beneficial properties. Due to highly-disseminated claims of antioxidant, antihypertensive and antiglycemic properties, BV is a known target for frauds and adulterations. For that matter, product authentication, certifying its origin (region or country) and thus the processing conditions, is becoming a growing concern. Striving for fraud reduction as well as quality and safety assurance, reliable analytical strategies to rapidly evaluate BV quality are very interesting, also from an economical point of view. This work employs silica plate laser desorption/ionization mass spectrometry (SP-LDI-MS) for fast chemical profiling of commercial BV samples with protected geographical indication (PGI) and identification of its adulterated samples with low-priced vinegars, namely apple, alcohol and red/white wines.

Estimated Disability-adjusted Life Years Averted By Long-term Provision Of Long Acting Contraceptive Methods In A Brazilian Clinic.

What is the contribution of the provision, at no cost for users, of long acting reversible contraceptive methods (LARC; copper intrauterine device [IUD], the levonorgestrel-releasing intrauterine system [LNG-IUS], contraceptive implants and depot-medroxyprogesterone [DMPA] injection) towards the disability-adjusted life years (DALY) averted through a Brazilian university-based clinic established over 30 years ago. Over the last 10 years of evaluation, provision of LARC methods and DMPA by the clinic are estimated to have contributed to DALY averted by between 37 and 60 maternal deaths, 315-424 child mortalities, 634-853 combined maternal morbidity and mortality and child mortality, and 1056-1412 unsafe abortions averted. LARC methods are associated with a high contraceptive effectiveness when compared with contraceptive methods which need frequent attention; perhaps because LARC methods are independent of individual or couple compliance. However, in general previous studies have evaluated contraceptive methods during clinical studies over a short period of time, or not more than 10 years. Furthermore, information regarding the estimation of the DALY averted is scarce. We reviewed 50 004 medical charts from women who consulted for the first time looking for a contraceptive method over the period from 2 January 1980 through 31 December 2012. Women who consulted at the Department of Obstetrics and Gynaecology, University of Campinas, Brazil were new users and users switching contraceptive, including the copper IUD (n = 13 826), the LNG-IUS (n = 1525), implants (n = 277) and DMPA (n = 9387). Estimation of the DALY averted included maternal morbidity and mortality, child mortality and unsafe abortions averted. We obtained 29 416 contraceptive segments of use including 25 009 contraceptive segments of use from 20 821 new users or switchers to any LARC method or DMPA with at least 1 year of follow-up. The mean (± SD) age of the women at first consultation ranged from 25.3 ± 5.7 (range 12-47) years in the 1980s, to 31.9 ± 7.4 (range 16-50) years in 2010-2011. The most common contraceptive chosen at the first consultation was copper IUD (48.3, 74.5 and 64.7% in the 1980s, 1990s and 2000s, respectively). For an evaluation over 20 years, the cumulative pregnancy rates (SEM) were 0.4 (0.2), 2.8 (2.1), 4.0 (0.4) and 1.3 (0.4) for the LNG-IUS, the implants, copper IUD and DMPA, respectively and cumulative continuation rates (SEM) were 15.1 (3.7), 3.9 (1.4), 14.1 (0.6) and 7.3 (1.7) for the LNG-IUS, implants, copper IUD and DMPA, respectively (P < 0.001). Over the last 10 years of evaluation, the estimation of the contribution of the clinic through the provision of LARC methods and DMPA to DALY averted was 37-60 maternal deaths; between 315 and 424 child mortalities; combined maternal morbidity and mortality and child mortality of between 634 and 853, and 1056-1412 unsafe abortions averted. The main limitations are the number of women who never returned to the clinic (overall 14% among the four methods under evaluation); consequently the pregnancy rate could be different. Other limitations include the analysis of two kinds of copper IUD and two kinds of contraceptive implants as the same IUD or implant, and the low number of users of implants. In addition, the DALY calculation relies on a number of estimates, which may vary in different parts of the world. LARC methods and DMPA are highly effective and women who were well-counselled used these methods for a long time. The benefit of averting maternal morbidity and mortality, child mortality, and unsafe abortions is an example to health policy makers to implement more family planning programmes and to offer contraceptive methods, mainly LARC and DMPA, at no cost or at affordable cost for the underprivileged population. This study received partial financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant # 2012/12810-4 and from the National Research Council (CNPq), grant #573747/2008-3. B.F.B., M.P.G., and V.M.C. were fellows from the scientific initiation programme from FAPESP. Since the year 2001, all the TCu380A IUD were donated by Injeflex, São Paulo, Brazil, and from the year 2006 all the LNG-IUS were donated by the International Contraceptive Access Foundation (ICA), Turku, Finland. Both donations are as unrestricted grants. The authors declare that there are no conflicts of interest associated with this study.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Sinonasal Disorders In Hematopoietic Stem Cell Transplantation.

hematopoietic stem cell transplantation (HSCT) is associated with more respiratory infections due to immunosuppression. this study aimed to verify the frequency of rhinosinusitis after HSCT, and the association between rhinosinusitis and chronic graft vs. host disease (GVHD) and type of transplantation, clinical treatment, surgical treatment, and survival. this was a retrospective study in a tertiary university hospital. A total of 95 patients with hematological diseases undergoing HSCT between 1996 and 2011 were selected. chronic myeloid leukemia was the most prevalent disease. The type of transplant most often performed was the allogenic type (85.26%). The frequency of rhinosinusitis was 36%, with no difference between the autologous and the allogenic types. Chronic GVHD occurred in 30% of patients. Patients with GVHD had a higher frequency and recurrence of rhinosinusitis, in addition to more frequent need for endoscopic sinusectomy and decreased overall survival. there was a higher frequency of rhinosinusitis in HSCT and GVHD. The type of transplant does not appear to predispose to the occurrence of rhinosinusitis. GVHD seems to be an aggravating factor and requires a more stringent treatment.

Guidelines On The Treatment Of Anemia Of Chronic Renal Failure Using Recombinant Human Erythropoietin: Associação Brasileira De Hematologia, Hemoterapia E Terapia Celular Guidelines Project: Associação Médica Brasileira - 2014.

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Onset Of Hypertension During Pregnancy Is Associated With Long-term Worse Blood Pressure Control And Adverse Cardiac Remodeling.

Up to 20% of women with hypertensive pregnancy disorders might persist with chronic hypertension. This study compared clinical and echocardiographic features between women whose hypertension began as hypertensive pregnancy disorders (PH group) and women whose diagnosis of hypertension did not occur during pregnancy (NPH group). Fifty PH and 100 NPH women were cross-sectionally evaluated by clinical, laboratory, and echocardiography analysis, and the groups were matched by duration of hypertension. PH exhibited lower age (46.6 ± 1.4 vs. 65.3 ± 1.1 years; P < .001), but higher systolic (159.8 ± 3.9 vs. 148.0 ± 2.5 mm Hg; P = .009) and diastolic (97.1 ± 2.4 vs. 80.9 ± 1.3 mm Hg; P < .001) blood pressure than NPH, although used more antihypertensive classes (3.4 ± 0.2 vs. 2.6 ± 0.1; P < .001). Furthermore, PH showed higher left ventricular wall thickness and increased prevalence of concentric hypertrophy than NPH after adjusting for age and blood pressure. In conclusion, this study showed that PH may exhibit worse blood pressure control and adverse left ventricular remodeling compared with NPH.

Hdl Levels And Oxidizability During Myocardial Infarction Are Associated With Reduced Endothelial-mediated Vasodilation And Nitric Oxide Bioavailability.

Acute phase response modifies high-density lipoprotein (HDL) into a dysfunctional particle that may favor oxidative/inflammatory stress and eNOS dysfunction. The present study investigated the impact of this phenomenon on patients presenting ST-elevation myocardial infarction (STEMI). Plasma was obtained from 180 consecutive patients within the first 24-h of onset of STEMI symptoms (D1) and after 5 days (D5). Nitrate/nitrite (NOx) and lipoproteins were isolated by gradient ultracentrifugation. The oxidizability of low-density lipoprotein incubated with HDL (HDLaoxLDL) and the HDL self-oxidizability (HDLautox) were measured after CuSO4 co-incubation. Anti-inflammatory activity of HDL was estimated by VCAM-1 secretion by human umbilical vein endothelial cells after incubation with TNF-α. Flow-mediated dilation (FMD) was assessed at the 30(th) day (D30) after STEMI. Among patients in the first tertile of admission HDL-Cholesterol (<33 mg/dL), the increment of NOx from D1 to D5 [6.7(2; 13) vs. 3.2(-3; 10) vs. 3.5(-3; 12); p = 0.001] and the FMD adjusted for multiple covariates [8.4(5; 11) vs 6.1(3; 10) vs. 5.2(3; 10); p = 0.001] were higher than in those in the second (33-42 mg/dL) or third (>42 mg/dL) tertiles, respectively. From D1 to D5, there was a decrease in HDL size (-6.3 ± 0.3%; p < 0.001) and particle number (-22.0 ± 0.6%; p < 0.001) as well as an increase in both HDLaoxLDL (33%(23); p < 0.001) and HDLautox (65%(25); p < 0.001). VCAM-1 secretion after TNF-a stimulation was reduced after co-incubation with HDL from healthy volunteers (-24%(33); p = 0.009), from MI patients at D1 (-23%(37); p = 0.015) and at D30 (-22%(24); p = 0.042) but not at D5 (p = 0.28). During STEMI, high HDL-cholesterol is associated with a greater decline in endothelial function. In parallel, structural and functional changes in HDL occur reducing its anti-inflammatory and anti-oxidant properties.

Digestive Peptidase Evolution In Holometabolous Insects Led To A Divergent Group Of Enzymes In Lepidoptera.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Surgical Treatment Of Type 2 Diabetes In Subjects With Mild Obesity: Mechanisms Underlying Metabolic Improvements.

This study aims to assess the clinical and physiological effects of Roux-en-Y gastric bypass (RYGBP) on type 2 diabetes associated with mild obesity (body mass index [BMI] 30-34.9 kg/m(2)) over 24 months postsurgery. In this prospective trial, 36 mildly obese subjects (19 males) with type 2 diabetes using oral antidiabetic drugs with (n = 24) or without insulin (n = 12) underwent RYGBP. Follow-up was conducted at baseline and 3, 6, 12, and 24 months postsurgery. The following endpoints were considered: changes in HbA1c, fasting glucose and insulin, antidiabetic therapy, BMI, oral glucose insulin sensitivity [OGIS, from meal tolerance test (MTT)], beta-cell secretory function [ΔCP(0-30)/ΔGlu(0-30) (ΔC-peptide/Δglucose ratio, MTT 0-30 min), disposition index (DI = OGIS [Symbol: see text] ΔCP(0-30)/ΔGlu(0-30)], glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) [incremental area under the curve (AUCi)], adiponectin, C-reactive protein, and lipids. All subjects achieved normal-to-overweight BMI after 3 months. Over 24 months, 31/36 (86 %) subjects presented HbA1c <7 % [complete and partial remission of diabetes in 9/36 (22 %) and 1/36 (3 %), respectively]. Since 3 months postsurgery, improvements were observed in OGIS [290 (174) to 373 (77) ml/min/m(2), P = 0.009], ΔCP(0-30)/ΔGlu(0-30) [0.24 (0.19) to 0.52 (0.34) ng/mg, P = 0.001], DI [7.16 (8.53) to 19.8 (15.4) (ng/mg) (ml/min/m(2)), P = 0.001], GLP-1 AUCi [0.56 (0.64) to 3.97 (3.86) ng/dl [Symbol: see text] 10 min [Symbol: see text] 103, P = 0.000], and GIP AUCi [30.2 (12.6) to 27.0 (20.2) ng/dl [Symbol: see text] 10 min [Symbol: see text] 103, P = 0.004]. At baseline and after 12 months, subjects with diabetes nonremission had longer diabetes duration, higher HbA1c, lower beta-cell secretory function, and higher first 30-min GIP AUCi, compared with those with remission. RYGBP improves the glucose metabolism in subjects with type 2 diabetes and mild obesity. This effect is associated with improvement of insulin sensitivity, beta-cell secretory function, and incretin secretion.

Association Of Single Nucleotide Polymorphisms In The Gene Encoding Glut1 And Diabetic Nephropathy In Brazilian Patients With Type 1 Diabetes Mellitus.

Mesangial cells subject to high extracellular glucose concentrations, as occur in hyperglycaemic states, are unable to down regulate glucose influx, resulting in intracellular activation of deleterious biochemical pathways. A high expression of GLUT1 participates in the development of diabetic glomerulopathy. Variants in the gene encoding GLUT1 (SLC2A1) have been associated to this diabetic complication. The aim of this study was to test whether polymorphisms in SLC2A1 confer susceptibility to diabetic nephropathy (DN) in Brazilian type 1 diabetes patients. Four polymorphisms (rs3820589, rs1385129, rs841847 and rs841848) were genotyped in a Brazilian cohort comprised of 452 patients. A prospective analysis was performed in 155 patients. Mean duration of follow-up was 5.6±2.4years and the incidence of renal events was 18.0%. The rs3820589 presented an inverse association with the prevalence of incipient DN (OR: 0.36, 95% CI: 0.16 - 0.80, p=0.01) and with progression to renal events (HR: 0.20; 95% CI: 0.03 - 0.70; p=0.009). AGGT and AGAC haplotypes were associated with the prevalence of incipient DN and the AGAC haplotype was also associated with the prevalence of established/advanced DN. In conclusion, rs3820589 in the SLC2A1 gene modulates the risk to DN in Brazilian patients with inadequate type 1 diabetes control.

Short-term Physiological Changes In Roots And Leaves Of Sugarcane Varieties Exposed To H2o2 In Root Medium.

The aim of this study was to evaluate the differential sensitivity of sugarcane genotypes to H2O2 in root medium. As a hypothesis, the drought tolerant genotype would be able to minimize the oxidative damage and maintain the water transport from roots to shoots, reducing the negative effects on photosynthesis. The sugarcane genotypes IACSP94-2094 (drought tolerant) and IACSP94-2101 (drought sensitive) were grown in a growth chamber and exposed to three levels of H2O2 in nutrient solution: control; 3mmolL(-1) and 80mmolL(-1). Leaf gas exchange, photochemical activity, root hydraulic conductance (Lr) and antioxidant metabolism in both roots and leaves were evaluated after 15min of treatment with H2O2. Although, root hydraulic conductance, stomatal aperture, apparent electron transport rate and instantaneous carboxylation efficiency have been reduced by H2O2 in both genotypes, IACSP94-2094 presented higher values of those variables as compared to IACSP94-2101. There was a significant genotypic variation in relation to the physiological responses of sugarcane to increasing H2O2 in root tissues, being root changes associated with modifications in plant shoots. IACSP94-2094 presented a root antioxidant system more effective against H2O2 in root medium, regardless H2O2 concentration. Under low H2O2 concentration, water transport and leaf gas exchange of IACSP94-2094 were less affected as compared to IACSP94-2101. Under high H2O2 concentration, the lower sensitivity of IACSP94-2094 was associated with increases in superoxide dismutase activity in roots and leaves and increases in catalase activity in roots. In conclusion, we propose a general model of sugarcane reaction to H2O2, linking root and shoot physiological responses.

A Rapid And Simple Capillary Electrophoresis Method For Indirect Determination Of The Biocide 2,2-dibromo-3-nitrilo-propionamide (dbnpa) In Cooling Waters.

A capillary zone electrophoresis (CE) method was developed for the determination of the biocide 2,2-dibromo-3-nitrilo-propionamide (DBNPA) in water used in cooling systems. The biocide is indirectly determined by CE measurement of the concentration of bromide ions produced by the reaction between the DBNPA and bisulfite. The relationship between the bromide peak areas and the DBNPA concentrations showed a good linearity and a coefficient of determination (R(2)) of 0.9997 in the evaluated concentration range of 0-75 μmol L(-1). The detection and quantification limits for DBNPA were 0.23 and 0.75 μmol L(-1), respectively. The proposed CE method was successfully applied for the analysis of samples of tap water and cooling water spiked with DBNPA. The intra-day and inter-day (intermediary) precisions were lower than 2.8 and 6.2%, respectively. The DBNPA concentrations measured by the CE method were compared to the values obtained by a spectrophotometric method and were found to agree well.

Chronic Continuous Exenatide Infusion Does Not Cause Pancreatic Inflammation And Ductal Hyperplasia In Non-human Primates.

In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67-positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67-positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67-positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30-positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a β-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates.