Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. Methods: Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. Results: In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2) = 0.33 and r(2) = 0.34 respectively, p < 0.0015). In patients with a low platelet count (<150 x 10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2) = 0.33 and r(2) = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05). Conclusions: There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count. (C) 2009 Elsevier Ltd. All rights reserved.

Early Increase in Myocardial Perfusion After Stem Cell Therapy in Patients Undergoing Incomplete Coronary Artery Bypass Surgery

Incomplete revascularization is associated with worse long-term outcomes. Autologous bone marrow cells (BMC) have recently been tested in patients with severe coronary artery disease. We tested the hypothesis that intramyocardial injection of autologous BMC increases myocardial perfusion in patients undergoing incomplete coronary artery bypass grafting (CABG). Twenty-one patients (19 men), 59 +/- 7 years old, with limiting angina and multivessel coronary artery disease (CAD), not amenable to complete CABG were enrolled. BMC were obtained prior to surgery, and the lymphomonocytic fraction separated by density gradient centrifugation. During surgery, 5 mL containing 2.1 +/- 1.3 x 10(8) BMC (CD34+ = 0.8 +/- 0.3%) were injected in the ischemic non-revascularized myocardium. Myocardial perfusion was assessed by magnetic resonance imaging (MRI) at baseline and 1 month after surgery. The increase in myocardial perfusion was compared between patients with < 50% (group A, n = 11) with that of patients with > 50% (group B, n = 10) of target vessels (stenosis a parts per thousand yenaEuro parts per thousand 70%) successfully bypassed. Injected myocardial segments included the inferior (n = 12), anterior (n = 7), and lateral (n = 2) walls. The number of treated vessels (2.3 +/- 0.8) was significantly smaller than the number of target vessels (4.2 +/- 1.0; P < 0.0001). One month after surgery, cardiac MRI showed a similar reduction (%) in the ischemic score of patients in group A (72.5 +/- 3.2), compared to patients in group B (78.1 +/- 3.2; P = .80). Intramyocardial injection of autologous BMC may help increase myocardial perfusion in patients undergoing incomplete CABG, even in those with fewer target vessels successfully treated. This strategy may be an adjunctive therapy for patients suffering from a more advanced (diffuse) CAD not amenable for complete direct revascularization.

NKX2.5 mutations in patients with non-syndromic congenital heart disease

Background: Cardiac development is a complex and multifactorial biological process. Heterozygous mutations in the transcription factor NKX2.5 are between the first evidence of a genetic cause for congenital heart defects in human beings. In this study, we evaluated the presence and frequency of mutations in the NKX2.5 gene on 159 unrelated patients with a diverse range of non-syndromic congenital heart defects (conotruncal anomalies, septal defects, left-sided lesions, right-sided lesions, patent ductus arteriosus and Ebstein`s anomaly). Methods: The coding region of the NKX2.5 locus was amplified by polymerase chain reaction and mutational analysis was performed using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. Results: We identified two distinct mutations in the NKX2.5 coding region among the 159 (1.26%) individuals evaluated. An Arg25Cys mutation was identified in a patient with Tetralogy of Fallot. The second mutation found was an Ala42Pro in a patient with Ebstein`s anomaly. Conclusions: The association of NKX2.5 mutations is present in a small percentage of patients with non-syndromic congenital heart defects and may explain only a few cases of the disease. Screening strategies considering the identification of germ-line molecular defects in congenital heart disease are still unwarranted and should consider other genes besides NKX2.5. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Causes of death and cardiovascular complications in adolescents and adults with congenitally malformed hearts: an autopsy study of 102 cases

Objectives: To identify the causes of death and main cardiovascular complications in adolescents and adults with congenitally malformed hearts. Design: Retrospective review of 102 necropsy reports from a tertiary centre obtained over a period of 19 years. Methods: The diagnosis, the operated or non-operated state of the main defect, the cause of death, and main complications were related to the age and gender. Other clinically relevant conditions, and identifiable sequels of previous diseases, were also noted. Results: The ages ranged from 15 to 69 years, with a mean of 31.1 and a median of 28 years, with no difference detected according to the gender. Of the patients, two-thirds had been submitted to at least one cardiac surgery. The mean age of death was significantly higher in non-operated patients (p = 0.003). The most prevalent cause of death in the whole group was related to recent surgery, found in one-third. From them, two-fifths corresponded to reoperations. Among the others, cardiac failure was the main terminal cause in another third, and the second cause was pulmonary thromboembolism in just over one-fifth, presenting a significant association with histopathological signs of pulmonary hypertension (p = 0.011). Infection was the cause of death in 7.8% of the patients, all previously operated. Acute infective endocarditis was present or was the indication for the recent surgery in one-tenth of the patients, this cohort having a mean age of 27.8 years. There was a statistically significant association between the occurrence of endocarditis and defects causing low pulmonary blood flow (p = 0.043). Conclusions: Data derived from necropsies of adults with congenital heart defects can help the multidisciplinary team refine both their diagnosis and treatment.

Role of Fos-related antigen 1 in the progression and prognosis of ductal breast carcinoma

Aims: Fos-related antigen 1 (Fra-1) is a member of the activator protein 1 (AP-1) transcription factor family. Our objective was to evaluate the role of Fra-1 expression in breast carcinoma progression and prognosis. Methods and results: Fra-1 expression was investigated by immunohistochemistry in two tissue microarrays containing, respectively, 85 ductal carcinoma in situ (DCIS) and 771 invasive ductal carcinoma (IDC) samples. Staining was observed in the nucleus and cytoplasm of the carcinomas, but only nuclear staining was considered to be positive. Fibroblasts associated with IDC were also Fra-1-positive. The frequency of Fra-1 positivity in IDC (22.8%) was lower than that in DCIS (42.2%). No association was found between Fra-1 and clinico-pathological variables in DCIS. In IDC, Fra-1 expression correlated with aggressive phenotype markers, including: high grade, oestrogen receptor negativity and human epidermal growth factor receptor 2 (HER-2) positivity (P = 0.001, 0.015 and 0.004, respectively), and marginally with the presence of metastasis (P = 0.07). Fra-1 was more frequently positive in basal-like (34%) and in HER-2-positive (38.5%) subtypes than in luminal subtypes. Fra-1 presence did not correlate with survival. Conclusions: A high frequency of Fra-1 in DCIS tumours may be associated with early events in breast carcinogenesis. Although Fra-1 expression correlated with features of a more aggressive phenotype in IDC, no relationship with overall survival was found.

Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumors: implications for tumor metastasis

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a beta (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further de-creased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Basilar artery occlusive disease in stroke survivors in a multiethnic population

Objectives: To describe clinical, radiological findings, and outcome in a multiethnic population of stroke survivors with basilar artery occlusive disease (BAOC). Methods: Forty patients with infarcts in the basilar artery (BA) territory, alive 30 days after the ictus, participated in the study. BA stenosis (>50%) or occlusion was shown by magnetic resonance or digital subtraction angiography in all patients. Demographical, clinical and radiological characteristics were described. Modified Rankin Scale (MRS) scores at 30 days and 6 months after the ischemic event were evaluated. Association between demographical, clinical, radiological features and outcome were analyzed with Chi-square and Fisher`s exact tests. MRS scores at 30 days and 6 months were compared with the Wilcoxon test. Results: Sixty percent of the patients were men, and 33% were Afro-Brazilian. Mean age was 55.8 +/- 12.9 years. Most (90%) had multiple vascular risk factors. Stroke was preceded by TIA in 48% of the patients, and 80% had a history of arterial hypertension. The most common neurological symptom was vertigo/dizziness (60%) and the sign, hemiparesis (60%). Most of the infarcts were located in the pons (85%) and the BA middle third was the most frequently affected segment (33%). BA occlusion occurred in 58% of the patients. More severe vascular occlusive lesions were present in Whites (p = 0.002) and in patients with involvement of the middle third of the BA (p = 0.021). Large-artery atherosclerosis was the most common stroke etiology (88%) and was more frequent in older patients (p < 0.001). Most patients were treated with anticoagulation. MRS scores improved significantly at 6 months (p < 0.001): at this time, 78% of the patients had MRS scores between 0 and 2. Conclusions: We observed different results compared with other series: greater proportion of Afro-descendents, higher frequency of atherosclerosis and BA occlusion. Rates of preceding TIAs and good outcome at 6 months were similar to previously published data. These results represent a step forward towards understanding BAOC in a multiethnic context. (C) 2009 Elsevier B.V. All rights reserved.

Concomitant expression of epithelial-mesenchymal transition biomarkers in breast ductal carcinoma: Association with progression

Epithelial to mesenchymal transition (EMT) is a process implicated in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. We determined the expression of some putative EMT biomarkers including E-cadherin, beta-catenin, zinc finger factor Snail (Snail), transforming growth factor beta 1 (TGF beta 1), TGF beta type II receptor (TBRII) and the HGF receptor (c-met) and their possible correlation to progression and overall survival in a series of breast ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC). Biomarkers were immunohistochemically determined in 55 IDC specimens from which 21 had lymph node metastases and in 95 DCIS specimens, 46 of these cases associated to invasive carcinoma, in a tissue microarray (TMA). Positive cytoplasmic staining of TGF beta 1 (78.2%), c-met (43.6%), Snail (34.5%), TBRII (100%), membranous E-cadherin (74.5%) and membranous/cytoplasmic beta-catenin (71%) were detected in the IDC samples. Metastatic lymph node samples displayed similar frequencies. A significant increase of c-met and TGF beta 1 positivity along DCIS to IDC progression was noted but only TGF beta 1 positivity was associated with presence of lymph node metastases and advanced stages in IDC. The evaluation of the other EMT markers in DCIS did not show differences in positivity rate as compared to invasive carcinomas. DCIS either pure or associated to IDC showed similar expression of the analyzed biomarkers. All the carcinomas exhibited positive expression of TBRII. Associations between the markers, determined by Spearman`s correlation coefficient, showed a significant association between TGF beta 1 and respectively E-cadherin, beta-catenin and cmet in DCIS cases, but in invasive carcinomas only cadherin and catenin were positively correlated. Kaplan-Meier survival curves revealed that none of the EMT biomarkers analyzed were correlated with survival, which was significantly determined only by clinical and hormone receptor parameters.

Improved detection of incipient vascular changes by a biotechnological platform combining post mortem MRI in situ with neuropathology

The histopathological counterpart of white matter hyperintensities is a matter of debate. Methodological and ethical limitations have prevented this question to be elucidated. We want to introduce a protocol applying state-of-the-art methods in order to solve fundamental questions regarding the neuroimaging-neuropathological uncertainties comprising the most common white matter hyperintensities [WMHs] seen in aging. By this protocol, the correlation between signal features in in situ, post mortem MRI-derived methods, including DTI and MTR and quantitative and qualitative histopathology can be investigated. We are mainly interested in determining the precise neuroanatomical substrate of incipient WMHs. A major issue in this protocol is the exact co-registration of small lesion in a tridimensional coordinate system that compensates tissue deformations after histological processing. The protocol is based on four principles: post mortem MRI in situ performed in a short post mortem interval, minimal brain deformation during processing, thick serial histological sections and computer-assisted 3D reconstruction of the histological sections. This protocol will greatly facilitate a systematic study of the location, pathogenesis, clinical impact, prognosis and prevention of WMHs. (C) 2009 Elsevier B.V. All rights reserved.

Assessment of factors that confound MRI and neuropathological correlation of human postmortem brain tissue

In spite of considerable technical advance in MRI techniques, the optical resolution of these methods are still limited. Consequently, the delineation of cytoarchitectonic fields based on probabilistic maps and brain volume changes, as well as small-scale changes seen in MRI scans need to be verified by neuronanatomical/neuropathological diagnostic tools. To attend the current interdisciplinary needs of the scientific community, brain banks have to broaden their scope in order to provide high quality tissue suitable for neuroimaging- neuropathology/anatomy correlation studies. The Brain Bank of the Brazilian Aging Brain Research Group (BBBABSG) of the University of Sao Paulo Medical School (USPMS) collaborates with researchers interested in neuroimaging-neuropathological correlation studies providing brains submitted to postmortem MRI in-situ. In this paper we describe and discuss the parameters established by the BBBABSG to select and to handle brains for fine-scale neuroimaging-neuropathological correlation studies, and to exclude inappropriate/unsuitable autopsy brains. We tried to assess the impact of the postmortem time and storage of the corpse on the quality of the MRI scans and to establish fixation protocols that are the most appropriate to these correlation studies. After investigation of a total of 36 brains, postmortem interval and low body temperature proved to be the main factors determining the quality of routine MRI protocols. Perfusion fixation of the brains after autopsy by mannitol 20% followed by formalin 20% was the best method for preserving the original brain shape and volume, and for allowing further routine and immunohistochemical staining. Taken to together, these parameters offer a methodological progress in screening and processing of human postmortem tissue in order to guarantee high quality material for unbiased correlation studies and to avoid expenditures by post-imaging analyses and histological processing of brain tissue.

Musculoskeletal Dysfunction and Pain in Adults with Asthma

Background. The mechanical alterations related to the overload of respiratory muscles observed in adults with persistent asthma might lead to the development of chronic alterations in posture, musculoskeletal dysfunction and pain; however, these changes remain poorly understood. Objective. This study aimed to assess postural alignment, muscle shortening and chronic pain in adults with persistent asthma. Methods. This cross-sectional and controlled study enrolled 30 patients with mild (n = 17) and severe ( n = 13) persistent asthma. Fifteen non-asthmatic volunteers were also assessed. Asthma was classified by the Global Initiative for Asthma (GINA) guidelines. Postural alignment and muscle shortening were evaluated by head and shoulder positions, chest wall mobility, and posterior ( trunk and lower limb) muscle flexibility. In addition, the measures used were previously tested for their reproducibility. Pain complaints were also assessed. Results. In comparison with non-asthmatic subjects, patients with mild or severe persistent asthma held their head and shoulders more forward and had lower chest wall expansion, decreased shoulder internal rotation, and decreased thoracic spine flexibility. Chronic lower thoracic, cervical, and shoulder pain was significantly increased in patients with mild or severe asthma compared with non-asthmatic subjects (p < 0.05). Conclusion. Adults with persistent asthma have musculoskeletal dysfunction and chronic pain that is independent of the severity of their disease but that might be related to their age at the onset of disease symptoms.

Effects of Aerobic Training on Airway Inflammation in Asthmatic Patients

MENDES, F. A. R., F. M. ALMEIDA, A. CUKIER, R. STELMACH, W. JACOB-FILHO, M. A. MARTINS, and C. R. F. CARVALHO. Effects of Aerobic Training on Airway Inflammation in Asthmatic Patients. Med. Sci. Sports Exerc., Vol. 43, No. 2, pp. 197-203, 2011. Purpose: There is evidence suggesting that physical activity has anti-inflammatory effects in many chronic diseases; however, the role of exercise in airway inflammation in asthma is poorly understood. We aimed to evaluate the effects of an aerobic training program on eosinophil inflammation (primary aim) and nitric oxide (secondary aim) in patients with moderate or severe persistent asthma. Methods: Sixty-eight patients randomly assigned to either control (CG) or aerobic training (TG) groups were studied during the period between medical consultations. Patients in the CG (educational program + breathing exercises; N = 34) and TG (educational program + breathing exercises + aerobic training; N = 34) were examined twice a week during a 3-month period. Before and after the intervention, patients underwent induced sputum, fractional exhaled nitric oxide (FeNO), pulmonary function, and cardiopulmonary exercise testing. Asthma symptom-free days were quantified monthly, and asthma exacerbation was monitored during 3 months of intervention. Results: At 3 months, decreases in the total and eosinophil cell counts in induced sputum (P = 0.004) and in the levels of FeNO (P = 0.009) were observed after intervention only in the TG. The number of asthma symptom-free days and (V) over dotO(2max) also significantly improved (P < 0.001), and lower asthma exacerbation occurred in the TG (P < 0.01). In addition, the TG presented a strong positive relationship between baseline FeNO and eosinophil counts as well as their improvement after training (r = 0.77 and r = 0.9, respectively). Conclusions: Aerobic training reduces sputum eosinophil and FeNO in patients with moderate or severe asthma, and these benefits were more significant in subjects with higher levels of inflammation. These results suggest that aerobic training might be useful as an adjuvant therapy in asthmatic patients under optimized medical treatment.

Endoscopic features in a model of multivisceral xenotransplantation

Introduction: Xenotransplantation and multivisceral transplantation are advanced therapeutic methods that still require a scientific basis. There are no experimental models of multivisceral transplantation available, particularly not the monitoring by endoscopy. Here, we describe the endoscopic features in a model of multivisceral xenotransplantation. Methods: The distal esophagus, stomach, intestine, colon, liver, pancreas, and the kidneys with a common vascular pedicle were harvested from rabbits and implanted in swine (group I, n = 9) or in rabbits (group II, n = 4). Endoscopy was performed in the stomach, jejunum, and ascending colon at four consecutive time points (immediate after surgery and 10, 90, and 180 min after reperfusion). Lesions were macroscopically graded as mild, moderate, and severe. Biopsies were taken following sacrifice at 180 min after reperfusion. Results: In group I, the stomach, jejunum, and colon manifested a progression of lesions with predominance of mild lesions after 10 min, mild to moderate lesions after 90 min, and moderate to severe lesions after 180 min. In animals from group II, endoscopy showed normal features at all time points after reperfusion. Histopathologic analysis confirmed the diagnosis of hyperacute rejection in group I. Grafts from group II animals presented normal or mild ischemic/reperfusion injury. Conclusion: All animals subjected to multivisceral xenotransplantation showed a progression of endoscopic lesions with time after transplantation, while animals subjected to allotransplantation showed no aberrations in endoscopy. We conclude that endoscopy is a useful tool in the assessment of hyperacute rejection of a xenograft.

Effects of Somatosensory Stimulation on Motor Function After Subacute Stroke

Background. Previous works showed potentially beneficial effects of a single session of peripheral nerve sensory stimulation (PSS) on motor function of a paretic hand in patients with subacute and chronic stroke. Objective. To investigate the influence of the use of different stimulus intensities over multiple sessions (repetitive PSS [RPSS]) paired with motor training. Methods. To address this question, 22 patients were randomized within the second month after a single hemispheric stroke in a parallel design to application of 2-hour RPSS at 1 of 2 stimulus intensities immediately preceding motor training, 3 times a week, for 1 month. Jebsen-Taylor test (JTT, primary endpoint measure), pinch force, Functional Independence Measure (FIM), and corticomotor excitability to transcranial magnetic stimulation were measured before and after the end of the treatment month. JTT, FIM scores, and pinch force were reevaluated 2 to 3 months after the end of the treatment. Results. Baseline motor function tests were comparable across the 2 RPSS intensity groups. JTT improved significantly in the lower intensity RPSS group but not in the higher intensity RPSS group at month 1. This difference between the 2 groups reduced by months 2 to 3. Conclusions. These results indicate that multiple sessions of RPSS could facilitate training effects on motor function after subacute stroke depending on the intensity of stimulation. It is proposed that careful dose-response studies are needed to optimize parameters of RPSS stimulation before designing costly, larger, double-blind, multicenter clinical trials.

Osteoblastlike Cell Adhesion on Titanium Surfaces Modified by Plasma Nitriding

Purpose: The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Materials and Methods: Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nit riding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. Results: There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Conclusions: Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion. INT J ORAL MAXILLOFAC IMPLANTS 2011;26:237-244