In vivo blockade of Ca(+2)-dependent nitric oxide synthases impairs expressions of L-selectin and PECAM-1

Interactions of leukocytes with endothelium play a role for the immune system modulated by endogenous agents, such as glucocorticoids and nitric oxide (NO). Glucocorticoids inhibit leukocyte-endothelial interactions whereas the role of NO is still controversial. In this study, the activity of Ca(+2)-dependent nitric oxide synthases was in vivo blocked in male Wistar rats by given L-NAME, 20 mg kg(-1) for 14 days dissolved in drinking water and expression of adhesion molecules involved in leukocyte-endothelial interactions was investigated. Expressions of L-selectin and PECAM-I in peripheral leukocytes and PECAM-1 in endothelial cells were reduced by L-NAME treatment. Only L-selectin expression was controlled at transcriptional levels. These effects were not dependent on endogenous glucocorticoids, as corticosterone levels were not altered in NAME-treated rats. Our results show that NO, produced at physiological levels, controls expression of constitutive adhesion molecules expressions in cell membranes by different mechanisms of action. Published by Elsevier Inc.

The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta(2) integrin-dependent pathway

Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. Transendothelial migration of leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 X 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyi-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. (C) 2009 Elsevier Ltd. All rights reserved.

In vivo hydroquinone exposure alters circulating neutrophil activities and impairs LPS-induced lung inflammation in mice

Hydroquinone (HQ) is an environmental contaminant which causes immune toxicity. In this study, the effects of exposure to low doses of HQ on neutrophil mobilization into the LPS-inflamed lung were investigated. Male Swiss mice were exposed to aerosolized vehicle (control) or 12.5, 25 or 50 ppm HQ (1 h/day for 5 days). One hour later, oxidative burst, cell cycle. DNA fragmentation and adhesion molecules expressions in circulating neutrophils were determined by flow cytometry, and plasma malondialdehyde (MDA) levels were measured by HPLC. Also, 1 h later the last exposures, inflammation was induced by LPS inhalation (0.1 mg/ml/10 min) and 3 h later, the numbers of leukocytes in peripheral blood and in the bronchoalveolar lavage fluid (BALF) were determined using a Neubauer chamber and stained smears; adhesion molecules expressed on lung microvessel endothelial cells were quantified by immunohistochemistry; myeloperoxidase (MPO) activity was measured in the lung tissue by colorimetric assay; and cytokines in the BALF were determined by ELISA. In vivo HQ exposure augmented plasma MDA levels and oxidative activity of neutrophils, but did not cause alterations in cell cycle and DNA fragmentation. Under these conditions, the number of circulating leukocytes was not altered, but HQ exposure reduced LPS-induced neutrophil migration into the alveolar space, as these cells remained in the lung tissue. The impaired neutrophil migration into BALF may not be dependent on reduced cytokines secretions in the BALF and lung endothelial adhesion molecules expressions. However, HQ exposure increased the expression of beta(2) and beta(3) integrins and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in neutrophils, which were not further enhanced by fMLP in vitro stimulation, indicating that HQ exposure activates circulating neutrophils, impairing further stimulatory responses. Therefore, it has been shown, for the first time, that neutrophils are target of lower levels of in vivo HQ exposure, which may be considered in host defense in infectious diseases. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats

In addition to reducing blood pressure, hydralazine can reduce the production of inflammatory cytokines and reduce the expression of leukocyte adhesion molecules. Differences in leukocyte behavior and leukocyte adhesion molecule expression in spontaneously hypertensive rats (SHR) compared to normotensive rats have been reported. However, whether hydralazine can reduce leukocyte migration in vivo in hypertension and in normotension remains unknown. To address this question, male SHR and Wistar rats were treated for 15 days with hydralazine at a dose of similar to 3.5 mg/kg or similar to 14 mg/kg in their drinking water. The numbers of rollers and adherent and migrated cells were determined by direct vital microscopy, and blood pressure was assessed by tail plethysmography. In addition, following treatment with the higher dose, immunohistochemistry was used to measure the expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cells, while flow cytometry was used to evaluate the expression of leukocyte CD18 and L-selectin. Hydralazine reduced leukocyte adherence and migration in SHR either at the higher, that reduced blood pressure levels, or lower dose, which did not reduce it. Reduced ICAM-1 expression might be involved in the reduced migration observed in SHR. In Wistar rats, only at the higher dose hydralazine reduced blood pressure levels and leukocyte migration. Reduced P-selectin expression might be involved. We therefore conclude that hydralazine reduces leukocyte migration by different mechanisms in SHR and Wistar rats, specifically by reducing ICAM-1 expression in the former and P-selectin expression in the latter. (c) 2008 Elsevier B.V. All rights reserved.

Amlodipine reduces the antimigratory effect of diclofenac in spontaneously hypertensive rats

Amlodipine, an antihypertensive drug, and diclofenac, an anti inflammatory drug, may generally be combined, particularly in elderly patients; therefore, the potential for their interaction is high. We aim to determine if amlodipine interferes with the antimigratory effect of diclofenac. For this, male spontaneously hypertensive rats (SHRs) were treated with either diclofenac (1 mg.kg(-1).d(-1), 15 d) alone or combined with amlodipine (10 mg.kg(-1).d(-1), 15 d). Leukocyte rolling, adherence, and migration were studied by intravital microscopy. Diclofenac did not change (180.0 +/- 2.3), whereas amlodipine combined (163.4 +/- 5.1) or not (156.3 +/- 4.3) with diclofienac reduced the blood pressure (BP) levels in SHR (183.1 +/- 4.4). Diclofenac and amlodipine reduced leukocyte adherence, migration, and ICAM-I expression, whereas only diclofenac reduced rolling leukocytes as well. Combined with amlodipine, the effect of the diclofenac was reduced. Neither treatment tested increased the venular shear rate or modified the venular diameters, number of circulating leukocytes, P-selectin, PECAM-1, L-selectin, or CD-18 expressions. No difference could be found in plasma concentrations of both drugs given alone or in association. In conclusion, amlodipine reduces leukocyte migration in SHR, reducing endothelial cell ICAM-1 expression. Amlodipine reduces the effect of the diclofenac, possibly by the same mechanism. A pharmacokinetic interaction as well as an effect on the other adhesion molecules tested could be discarded.

Reduced allergic lung inflammation in rats following formaldehyde exposure: Long-term effects on multiple effector systems

Clinical and experimental evidences show that formaldehyde (FA) exposure has an irritant effect on the upper airways. As being an indoor and outdoor pollutant, FA is known to be a causal factor of occupational asthma. This study aimed to investigate the repercussion of FA exposure on the course of a lung allergic process triggered by an antigen unrelated to FA. For this purpose, male Wistar rats were subjected to FA inhalation for 3 consecutive days (1%, 90-min daily), subsequently sensitized with ovalbumin (OVA)-alum via the intraperitoneal route, and 2 weeks later challenged with aerosolized OVA. The OVA challenge in rats after FA inhalation (FA/OVA group) evoked a low-intensity lung inflammation as indicated by the reduced enumerated number of inflammatory cells in bronchoalveolar lavage as compared to FA-untreated allergic rats (OVA/OVA group). Treatment with FA also reduced the number of bone marrow cells and blood leukocytes in sensitized animals challenged with OVA, which suggests that the effects of FA had not been only localized to the airways. As indicated by passive cutaneous anaphylactic reaction, FA treatment did not impair the anti-OVA IgE synthesis, but reduced the magnitude of OVA challenge-induced mast cell degranulation. Moreover, FA treatment was associated to a diminished lung expression of PECAM-1 (platelet-endothelial cell adhesion molecule 1) in lung endothelial cells after OVA challenge and an exacerbated release of nitrites by BAL-cultured cells. Keeping in mind that rats subjected solely to either FA or OVA challenge were able to significantly increase the cell influx into lung, our study shows that FA inhalation triggers long-lasting effects that affect multiple mediator systems associated to OVA-induced allergic lung such as the reduction of mast cells activation, PECAM-1 expression and exacerbation of NO generation, thereby contributing to the decrease of cell recruitment after the OVA challenge. In conclusion, repeated expositions to air-borne FA may impair the lung cell recruitment after an allergic stimulus, thereby leading to a non-responsive condition against inflammatory stimuli likely those where mast cells are involved. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Comparison of three vascular endothelial markers in the evaluation of microvessel density in breast cancer

Purpose: To evaluate the microvessel density by comparing the performance of anti-factor VIII-related antigen, anti-CD31 and, anti-CD34 monoclonal antibodies in breast cancer. Methods: Twenty-three postmenopausal women diagnosed with Stage II breast cancer submitted to definitive surgical treatment were evaluated. The monoclonal antibodies used were anti-factor VIII, anti-CD31 and anti-CD34. Microvessels were counted in the areas of highest microvessel density in ten random fields (200 x). The data were analyzed using the Kruskal-Wallis nonparametric test (p < 0.05). Results: Mean microvessel densities with anti-factor VIII, anti-CD31 and anti-CD34 were 4.16 +/- 0.38, 4.09 +/- 0.23 and 6.59 +/- 0.42, respectively. Microvessel density as assessed by anti-CD34 was significantly greater than that detected by anti-CD31 or anti-factor VIII (p < 0.0001). There was no statistically significant difference between anti-CD31 and anti-factor VIII (p = 0.4889). Conclusion: The density of stained microvessels was greater and staining was more intense with anti-CD34 compared to anti-CD31 and anti-factor VII-related antigen.

Polymorphisms in genes involved in neurodevelopment may be associated with altered brain morphology in schizophrenia: Preliminary evidence

An abnormality in neurodevelopment is one of the most robust etiologic hypotheses in schizophrenia (SZ). There is also strong evidence that genetic factors may influence abnormal neurodevelopment in the disease. The present study evaluated in SZ patients, whose brain structural data had been obtained with magnetic resonance imaging (MRI), the possible association between structural brain measures, and 32 DNA polymorphisms,located in 30 genes related to neurogenesis and brain development. DNA was extracted from peripheral blood cells of 25 patients with schizophrenia, genotyping was performed using diverse procedures, and putative associations were evaluated by standard statistical methods (using the software Statistical Package for Social Sciences - SPSS) with a modified Bonferroni adjustment. For reelin (RELN), a protease that guides neurons in the developing brain and underlies neurotransmission and synaptic plasticity in adults, an association was found for a non-synonymous polymorphism (Va1997Leu) with left and right ventricular enlargement. A putative association was also found between protocadherin 12 (PCDH12), a cell adhesion molecule involved in axonal guidance and synaptic specificity, and cortical folding (asymmetry coefficient of gyrification index). Although our results are preliminary, due to the small number of individuals analyzed, such an approach could reveal new candidate genes implicated in anomalous neurodevelopment in schizophrenia. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Role of dietary fats in modulating cardiometabolic risk during moderate weight gain : a randomized double-blind overfeeding trial (LIPOGAIN study)

BACKGROUND: Whether the type of dietary fat could alter cardiometabolic responses to a hypercaloric diet is unknown. In addition, subclinical cardiometabolic consequences of moderate weight gain require further study. METHODS AND RESULTS: In a 7-week, double-blind, parallel-group, randomized controlled trial, 39 healthy, lean individuals (mean age of 27±4) consumed muffins (51% of energy [%E] from fat and 44%E refined carbohydrates) providing 750 kcal/day added to their habitual diets. All muffins had identical contents, except for type of fat; sunflower oil rich in polyunsaturated fatty acids (PUFA diet) or palm oil rich in saturated fatty acids (SFA diet). Despite comparable weight gain in the 2 groups, total: high-density lipoprotein (HDL) cholesterol, low-density lipoprotein:HDL cholesterol, and apolipoprotein B:AI ratios decreased during the PUFA versus the SFA diet (-0.37±0.59 versus +0.07±0.29, -0.31±0.49 versus +0.05±0.28, and -0.07±0.11 versus +0.01±0.07, P=0.003, P=0.007, and P=0.01 for between-group differences), whereas no significant differences were observed for other cardiometabolic risk markers. In the whole group (ie, independently of fat type), body weight increased (+2.2%, P<0.001) together with increased plasma proinsulin (+21%, P=0.007), insulin (+17%, P=0.003), proprotein convertase subtilisin/kexin type 9, (+9%, P=0.008) fibroblast growth factor-21 (+31%, P=0.04), endothelial markers vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (+9, +5, and +10%, respectively, P<0.01 for all), whereas nonesterified fatty acids decreased (-28%, P=0.001). CONCLUSIONS: Excess energy from PUFA versus SFA reduces atherogenic lipoproteins. Modest weight gain in young individuals induces hyperproinsulinemia and increases biomarkers of endothelial dysfunction, effects that may be partly outweighed by the lipid-lowering effects of PUFA. CLINICAL TRIAL REGISTRATION URL: http://ClinicalTrials.gov. Unique identifier: NCT01427140.

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

Estudo das características físico-químicas e biológicas pela adesão de osteoblastos em superfícies de titânio modificadas pela nitretação em plasma

SILVA, J. S. P. Estudo das características físico-químicas e biológicas pela adesão de osteoblastos em superfícies de titânio modificadas pela nitretação em plasma. 2008. 119 f. Tese (Doutorado) - Faculdade de Medicina, Universidade de São Paulo. São Paulo, 2008.

Avaliação do potencial antiinflamatório, anticoagulante e hemorrágico de heparinóides presentes em "Artemia franciscana"

Heparan sulfate (HS) and Heparin (Hep) glycosaminoglycans (GAGs) are heterogeneous and highly charged polysaccharides. HS is structurally related to Hep but is much less substituted with sulfo groups than heparin and has a more varied structure (or sequence). Because of structural similiarities between these two polymers, they have been described together as heparinoids . Both chains bind a variety of proteins and mediate various physiologically important processes including, blood coagulation, cell adhesion and growth factor regulation. Heparinoids with structural characteristics similar to these described from HS and/or Hep from mammalian tissues have been isolated from different species of invertebrates, although only a few heparinoids from unusual sources have been characterized. The present study describes the presence of unusual heparinoids population from Artemia franciscana, isolated after proteolysis and fractionation by ion exchange resin and named, F-3.0M. The study model in vivo were hemostasis (rat tail scarification) and inflamatoty activity. The tests in vitro were used for coagulations assays (PT and APTT). The analyse of the heparinoids eluted with 3,0M NaCl showed electrophoretic migration in different buffer systems a single band with a behaviour intermediate between those of mammalian HEP and HS. The main products obtained from Artemia heparinoids after enzymatic degradation with heparitinases I and II from F. heparinum were N-sulphated disaccharides (∆U-GlcNS,6S/ ∆U,2S-GlcNS and ∆U-GlcNS) and N-acetylated disaccharides (∆U, GlcNAc). This heparinoid had a lower hemorrhagic effect (400μg/ml) when compared to unfractiionated heparins(25μg/ml).The results also suggest a negligible APTT activity of this heparinoid (62.2s). No action was observed on PT indicating that F-3.0M haven t action on the extrinsic pathway. The results showed that the fraction F- 3.0M have inhibitory effect on migration of leukocytes, 64.5% in the concentration of 10 μg/ml (P<0.001). The search for new heparin and/or heparan sulphates analogs devoid of anticoagulant activity is an atractive alternative and may open up a wide variety of new therapeutic applications

Adesão, proliferação e genotoxicidade celular de celulose bacteriana modificada por plasma

Bacterial cellulose (BC) has a wide range of potential applications, namely as temporary substitute skin in the treatment of skin wounds, such as burns, ulcers and grafts. Surface properties determine the functional response of cells, an important factor for the successful development of biomaterials. This work evaluates the influence of bacterial cellulose surface treatment by plasma (BCP) on the cellular behavior and its genotoxicity potential. The modified surface was produced by plasma discharge in N2 and O2 atmosphere, and the roughness produced by ion bombardment characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell adhesion, viability and proliferation on BCP were analysed using crystal violet staining and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) method. Genotoxicity was evaluated using the comet and cytokinesis block micronucleus assay. The results show that the plasma treatment changed surface roughness, producing an ideal cell attachment, evidenced by more elongated cell morphology and improved proliferation. The excellent biocompatibility of BCP was confirmed by genotoxicity tests, which showed no significant DNA damage. The BCP has therefore great potential as a new artificial implant