Aline Mendes

Distribution of sulfated glycosaminoglycans in the animal kingdom: widespread occurrence of heparin-like compounds in invertebrates

Sulfated glycosaminoglycans were isolated from 23 species of 13 phyla of invertebrates and characterized by their electrophoretic migration in three different buffer systems coupled with enzymatic degradation using bacterial heparinase, heparitinases and chondroitinase AC. Heparan sulfate is a ubiquitous compound present in all species analyzed whereas chondroitin sulfate was present in 20 species and heparin-like compounds in 12 species of the invertebrates. The heparin-like compounds were purified from the echinoderm Mellita quinquisperforata (sand dollar) and the crustacean Ucides cordatus (crab) with anticoagulant activities of 60 and 52 IU/mg, respectively. Degradation of these heparins with heparinase produced significant amounts of the trisulfated disaccharide typical of mammalian heparins. This was confirmed by 13C-NMR spectroscopy of the crab heparin. An updated phylogenetic tree of the distribution of sulfated glycosaminoglycans in the animal kingdom is also presented.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.

Comparison of practical methods for urinary glycosaminoglycans and serum hyaluronan with clinical activity scores in patients with Graves' ophthalmopathy

objective  Immunosuppressive treatment of Graves’ opthalmopathy (GO) should be restricted to patients with active eye disease, but assessing disease activity is difficult. Several methods to evaluate GO activity have been introduced, but none of them is satisfactory. Glycosaminoglycans (GAGs) are complex polysaccharides that participate on the pathogenesis of GO and attempts to correlate its local increase to urinary GAGs (uGAGs) or serum hyaluronan (sHA) have been made, but the available techniques are labourious, time‐consuming and difficult for routine use. The aim of the present study is to develop practical and simple methods for uGAGs and sHA and compare them to the activity and severity of thyroid‐associated ophthalmopathy.

Objective Improvement in Nasal Congestion and Nasal Hyperreactivity with Use of Nasal Steroids in Persistent Allergic Rhinitis

Objective Our objective was to evaluate the action of topical nasal steroid in nasal congestion and nasal hyper-reactivity in children and adolescents with persistent allergic rhinitis. Methods Twenty atopic children and adolescents (6 to 18 years) with moderate-to-severe persistent allergic rhinitis (PAR) were treated with mometasone furoate (100 μg once a day) for 21 days. At the beginning and end of treatment, the following were recorded: a) nasal symptoms score; b) several parameters of nasal congestion measured by acoustic rhinometry (SRE 2000 Rhinometrics); c) degree of nasal hyper-reactivity to histamine (concentration of histamine necessary to induce at least 100% increase in total nasal resistance during nasal provocation test). Data were compared with those from 20 controls. Results Compared to controls, PAR patients had significantly higher score of symptoms, as well as higher degree of nasal hyper-reactivity and lower nasal volumes. After treatment, there was a significant decrease in the mean nasal symptoms score (8.0 versus 3.8; p < 0.001) and in the nasal hyper-reactivity (histamine concentration: 0.72 mg/ml versus 2.60 mg/ml; p < 0.001). Congestion improvement was observed by the increase in all acoustic rhinometry parameters. Among all studied volumes, the volume in the segment between 2 and 5 cm showed the highest mean increase (19.8%). Conclusions Treatment with topical nasal steroid objectively reduced nasal congestion and nasal histamine hyper-reactivity in children and adolescents with PAR.

Long-term effects for acute phase myocardial infarct VEGF165 gene transfer cardiac extracellular matrix remodeling

Background. Cardiac remodeling is ultimately regulated by components of the extracellular matrix (ECM). We investigated the important role that growth factors play in the regulation of ECM remodeling that occurs as a consequence of myocardium damage. Methods and results. Rats were submitted to the ligation of the left anterior coronary artery and pcDNA3-vascular endothelial growth factor (VEGF)165 was immediately injected intramyocardially in the treated group. The animals were divided into large size myocardium infarction (LMI) and small size myocardium infarction, with or without gene transfer. The plasmid-containing DNA encoding VEGF165 was injected into the cardiac muscle and its effect was observed on the ECM components. Glycosaminoglycans were identified and quantified by agarose gel based electrophoresis and ELISA as well as immunocytochemistry to examine specific cathepsin B, heparanase, and syndecan-4 changes. The amounts of hyaluronic acid (HA; p < 0.005), DS, chondroitin sulfate, and heparan sulfate (p < 0.001) were significantly increased in the LMI treated group in comparison to the other groups, which correlates with the decrease in the expression of heparanase. A decrease in the molecular mass of HA was found in the scar tissue of treated group. Conclusions. The data obtained strongly support the idea that changes in the ECM and its components are important determinants of cardiac remodeling after myocardium infarct and may be essential for inflammatory response and attempt to stabilize the damage and provide a compensatory mechanisms to maintain cardiac output since the ECM components analyzed are involved with angiogenesis, cell proliferation and differentiation.

A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities.

Avaliações objetiva e subjetiva da obstrução nasal em crianças e adolescentes com rinite alérgica

OBJECTIVE To correlate objective assessment of nasal obstruction, as measured by acoustic rhinometry (volume of the first 5 cm of the nasal cavity) and active anterior rhinomanometry (total nasal airway resistance), with its subjective evaluation (obstruction scores). METHOD Thirty patients, aged 7 to 18 years, with persistent allergic rhinitis and thirty controls were enrolled. The obstruction score was reported for the whole nasal cavity and for each nostril separately. The three variables were measured at baseline and after induction of nasal obstruction. RESULTS There were significant and negative correlations between resistance and nasal volume in all groups and scenarios, except for the most obstructed nostril, in the control group, post-obstruction. For the whole nasal cavity, there was no significant correlation between objective and subjective variables except between score and total nasal cavity volume in the control group, post-obstruction. Regarding the most obstructed nostril, we found a significant negative correlation between score and resistance and a significant positive correlation between score and volume for the total group at baseline. There were no clear differences in the correlation coefficients found in patients and controls. The correlation coefficients did not change after induction of nasal obstruction. CONCLUSIONS Objective assessment of nasal obstruction did not correlate significantly with subjective evaluation for the nasal cavity as a whole, but there was a correlation for unilateral assessments. There was correlation between the objective evaluations. Allergic rhinitis and acute induction of nasal obstruction did not affect the correlation between objective and subjective assessments of nasal obstruction. Addition of an objective method for evaluation of nasal obstruction could be useful in the research setting; if no such method can be used, each nostril should be evaluated separately.

Concentration of Hyaluronic Acid in Human Vocal Folds in Young and Old Subjects

Objective. To compare and evaluate concentrations of hyaluronic acid (HA) in normal vocal folds of both young and elderly people of both sexes. Study Design. Experimental study. Setting. The research was carried out at the ENT, Head and Neck Surgery Department and the Pharmacology Institute of São Paulo Federal University. Subjects and Methods. Enzyme-linked immunosorbent assay plates coated with biotin-conjugated binding protein and streptavidin-europium conjugate were sequentially incubated with samples from the middle portion of the cover, vocal ligament, and vocal muscle of 19 young and 20 elderly subjects of both sexes. After the release of europium in an enhancement solution, final fluorescence was measured using a fluorometer. Results. Statistically significant comparisons: (1) all subject groups combined exhibited a higher HA concentration in the cover and ligament compared with the muscle samples; (2) women subjects had a significantly lower HA concentration in muscle compared with the ligament, and male subjects showed significantly lower HA in muscle compared with the cover; and (3) both genders of young subjects showed a higher HA concentration in ligament samples compared with the muscle. Conclusion. HA concentrations are higher in the cover and ligament compared with the muscle in both genders and age groups, and there is a higher HA concentration in ligament compared with the muscle of young subjects of both genders. HA levels in the cover samples of younger women showed great variability that may relate to ovarian hormone levels, reflecting different phases of the menstrual cycle.

Correlação entre a resistência nasal e diferentes parâmetros da rinometria acústica em crianças e adolescentes com e sem rinite alérgica

UNLABELLED Acoustic rhinometry and rhinomanometry are important tests used to assess nasal function. The degree to which the parameters of these tests are correlated is yet to be established. OBJECTIVE This paper aimed to study the correlations between nasal resistance (NR) and acoustic rhinometry parameters in children and adolescents with allergic rhinitis and controls. METHOD Twenty patients with allergic rhinitis and 20 controls were enrolled. NR, volumes (V4, V5, V2-5), and minimal cross-sectional areas (MC1, MC2) were measured in three moments: baseline, after induction of nasal obstruction and after topical decongestant administration. RESULTS Patients with allergic rhinitis had significant correlation between NR and all volumes (V5: r = -0.60) and with MC2. Among controls, MC1 was the parameter with the strongest correlation with NR at baseline (r = -0.53) and after decongestant administration. In the combined analysis, V5 had the highest correlation coefficients at baseline (r = -0.53), after obstruction (r = -0.58) and after decongestant (r = -0.46). CONCLUSIONS Our data showed that NR and acoustic rhinometry parameters have negative and significant correlations. Nasal volumes are, in general, better correlated than minimal cross-sectional areas. V5 was the parameter with the highest correlation in the rhinitis group and in the combined analysis.