Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration.

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6 nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 µmol/kg significantly inhibited and at 1 µmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

Spatial linear navigation: is vision necessary?

In order to analyze spatial linear navigation through a task of self-controlled reproduction, healthy participants were passively transported on a mobile robot at constant velocity, and then had to reproduce the imposed distance of 2-8m in two conditions: "with vision" and "without vision". Our hypothesis was that the reproduction of distances would be longer with than without visual information. Indeed, with visual information the reproduction of all distances was overshot. In the "without vision" condition the reproduced distances were quite close to the imposed ones, but only for the shortest distances (2 and 4m) as the longest ones were clearly undershot. With vision the reproduction error was less than 10% for all distances; however the error could be smaller without vision at short distances, and therefore vision was not necessary.

A simple circuit for cerebral perfusion during cardiopulmonary bypass surgery of the ascending aorta and the aortic arch.

INTRODUCTION: A circuit was developed to allow for rapid reaction to the needs of perfusion during extracorporeal circulation (ECC) in surgery of the aortic arch and ascending aorta. METHOD: From January 2008 through January 2010, a home-designed circuit was used on 30 patients with aortic dissection who underwent surgery to replace the ascending aorta and the aortic arch and, in some cases, the aortic valve and re-implant of the coronary arteries using Bentall's technique.

Association of increased CCL5 and CXCL7 chemokine expression with neutrophil activation in severe stable COPD.

BACKGROUND: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. OBJECTIVES: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I-IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. METHODS: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). RESULTS: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2-15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. CONCLUSION: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients.

There are increased numbers of activated T lymphocytes in the bronchial mucosa of stable chronic obstructive pulmonary disease (COPD) patients. T helper type 17 (Th17) cells release interleukin (IL)-17 as their effector cytokine under the control of IL-22 and IL-23. Furthermore, Th17 numbers are increased in some chronic inflammatory conditions. To investigate the expression of interleukin (IL)-17A, IL-17F, IL-21, IL-22 and IL-23 and of retinoic orphan receptor RORC2, a marker of Th17 cells, in bronchial biopsies from patients with stable COPD of different severity compared with age-matched control subjects. The expression of IL-17A, IL-17F, IL-21, IL-22, IL-23 and RORC2 was measured in the bronchial mucosa using immunohistochemistry and/or quantitative polymerase chain reaction. The number of IL-22(+) and IL-23(+) immunoreactive cells is increased in the bronchial epithelium of stable COPD compared with control groups. In addition, the number of IL-17A(+) and IL-22(+) immunoreactive cells is increased in the bronchial submucosa of stable COPD compared with control non-smokers. In all smokers, with and without disease, and in patients with COPD alone, the number of IL-22(+) cells correlated significantly with the number of both CD4(+) and CD8(+) cells in the bronchial mucosa. RORC2 mRNA expression in the bronchial mucosa was not significantly different between smokers with normal lung function and COPD. Further, we report that endothelial cells express high levels of IL-17A and IL-22. Increased expression of the Th17-related cytokines IL-17A, IL-22 and IL-23 in COPD patients may reflect their involvement, and that of specific IL-17-producing cells, in driving the chronic inflammation seen in COPD.

Microscopic characterization of teeth of pacas bred in captivity (Agouti paca, Linnaeus, 1766).

The microscopic description of the teeth of pacas (Agouti paca) bred in captivity was developed for providing biological data on one of the largest American wild rodents, as not many references exist in the literature about this species. Two newborn males, two adult males (9 and 72 months old), one newborn female and two adult females (30 and 54 months old) were used after death due to fights, neonatal cannibalism or unknown causes. Animals were radiographed, and their teeth were extracted and put on an acrylic resin block, cut on a diamond-like disc microtome and diaphanized. It was noted that enamel surrounds the coronary dentine and projects to the root region, besides being present as internal laminae, arranged in a parallel way and in the vestibulolingual direction. The dentine is located between the enamel laminae and surrounds the pulp horns. The cementum is located internal to the enamel laminae. From scanning electronic microscopy, we find that the enamel is the outer element on the vestibular surface, and it is in direct contact with the dentine. On the lingual surface, the cementum and dentine are the outer elements.

Borderline HER-2 breast cancer cases: histochemical versus real-time PCR analysis and impact of different cut-off values.

Seventy-one cases that had resulted borderline for HER-2 protein expression at conventional immunohistochemical assay (2+) were assessed for HER-2 gene amplification by real-time PCR and by FISH in accordance with the manufacturer's recommendations (gene amplification with ratio >or=2 in both methods). Thirty-three out of 71 cases (47%) resulted amplified at real-time PCR analysis, whereas 15 cases resulted positive at FISH (21%). Apparently, PCR was more sensitive than FISH in HER-2 determination, only 10 cases resulting amplified in both tests. When the mean ratio value obtained in all PCR experiments was adopted as threshold in determining HER-2 gene amplification, the apparent sensitivity of PCR was reduced but correlation between PCR and FISH results was dramatically increased. Furthermore, when the mean PCR ratio value observed in the FISH-positive group was chosen as threshold, the best agreement between PCR and FISH results was achieved. Therefore, we found that the proposed threshold ratio value of >or=2 is not accurate in separating HER-2 amplified and non-amplified cases. We suggest that the threshold ratio value in PCR tests should be determined in each laboratory using FISH controlled cases. Finally, above certain in-lab generated threshold values, PCR might be proposed as a highly predictive positive test in HER-2 assessment.

One second interval production task during post-rotatory sensation.

Temporal intervals production of one second was found to be more variable during self-motion compared to no motion situations. Moreover, the temporal intervals production rhythm during self-motion deceleration decreased whereas it increased during self-motion acceleration, whatever the direction of motion. As somatosensory cues were not excluded in this previous experiment, we now examined whether the same temporal perturbation would occur without variable somatosensory information. In order to isolate the contribution of the vestibular system from that of the somatosensory system, the participants were required to perform a one second temporal interval production task (pressing a button each second) during the post-rotatory illusion following self-rotation. The intervals produced during the vestibular illusion were compared to those produced before the imposed rotation and during self-motion. The production regularity was affected as the temporal intervals were more variable with vestibular stimulation (real and illusory self-motion) than without. Furthermore, during post-rotatory illusion, the production rhythm decreased along the trial, as it was observed during self-motion deceleration. These findings suggest that vestibular stimulation (even vestibular illusion) impaired time estimation.

Anatomoradiographic description of the teeth of pacas bred in captivity (Agouti paca, Linnaeus, 1766).

Two newborn males, two adult males 9 and 72 months old, one newborn female and two adult females of 30 and 54 months old pacas were used. Animals were radiographed on lateral recumbency, teeth were extracted and the vestibulolingual and mesiodistal lengths were achieved at the occlusal surface, besides the longitudinal length. The lower incisors presented greater length in relation to the other ones and sinuous shape; the upper incisors, relatively smaller, are practically straight. There is no canine tooth, and after a great diastema there is one premolar and three molars in each hemiarcade. In adult pacas, the enamel of incisors presents yellowish colour, mainly in the labial surface; in the premolars and molars, the enamel is present as internal sheets disposed nearly in a parallel way and in vestibulolingual direction, through practically all the dental length, which provides several joined teeth aspect.

Influence of cervical preflaring on apical file size determination.

AIM: To investigate the influence of cervical preflaring with different instruments (Gates-Glidden drills, Quantec Flare series instruments and LA Axxess burs) on the first file that binds at working length (WL) in maxillary central incisors. METHODOLOGY: Forty human maxillary central incisors with complete root formation were used. After standard access cavities, a size 06 K-file was inserted into each canal until the apical foramen was reached. The WL was set 1 mm short of the apical foramen. Group 1 received the initial apical instrument without previous preflaring of the cervical and middle thirds of the root canal. Group 2 had the cervical and middle portion of the root canals enlarged with Gates-Glidden drills sizes 90, 110 and 130. Group 3 had the cervical and middle thirds of the root canals enlarged with nickel-titanium Quantec Flare series instruments. Titanium-nitrite treated, stainless steel LA Axxess burs were used for preflaring the cervical and middle portions of root canals from group 4. Each canal was sized using manual K-files, starting with size 08 files with passive movements until the WL was reached. File sizes were increased until a binding sensation was felt at the WL, and the instrument size was recorded for each tooth. The apical region was then observed under a stereoscopic magnifier, images were recorded digitally and the differences between root canal and maximum file diameters were evaluated for each sample. RESULTS: Significant differences were found between experimental groups regarding anatomical diameter at the WL and the first file to bind in the canal (P < 0.01, 95% confidence interval). The major discrepancy was found when no preflaring was performed (0.151 mm average). The LA Axxess burs produced the smallest differences between anatomical diameter and first file to bind (0.016 mm average). Gates-Glidden drills and Flare instruments were ranked in an intermediary position, with no statistically significant differences between them (0.093 mm average). CONCLUSIONS: The instrument binding technique for determining anatomical diameter at WL is not precise. Preflaring of the cervical and middle thirds of the root canal improved anatomical diameter determination; the instrument used for preflaring played a major role in determining the anatomical diameter at the WL. Canals preflared with LA Axxess burs created a more accurate relationship between file size and anatomical diameter.

CCR5 expression and CC chemokine levels in idiopathic pulmonary fibrosis.

CC chemokines play an important role in the pathogenetic mechanisms of interstitial lung disease, while a downregulation of CC chemokine receptor (CCR)5 in the fibrotic stages of sarcoidosis has been observed. To evaluate the involvement of CC chemokines and the expression of CCR5 in idiopathic pulmonary fibrosis (IPF) and, more specifically, in usual interstitial pneumonia, 35 subjects were studied. CC chemokine ligand (CCL)2, CCL3 and CCL4 levels were measured in the bronchoalveolar lavage fluid (BALF) of 18 nonsmoker control subjects and 17 patients affected by IPF. CCR5 expression was evaluated in alveolar macrophages and lymphocytes. The BALF levels of all chemokines were significantly increased in IPF: median (range) CCL3 1.6 (1.0-11.1) versus 1.2 (0.0-3.8) pg x mL(-1); CCL4 6.2 (1.3-96.0) versus 3.4 (0.3-6.8) pg x mL(-1); and CCL2 60.1 (16.7-251.3) versus 4.6 (0.5-119.4) pg x mL(-1). CCL2 levels correlated negatively with the carbon monoxide diffusing capacity of the lung (D(L,CO)) and arterial oxygen tension. CCR5 expression was significantly reduced in lymphocytes from IPF compared with controls. The CC chemokines investigated are involved in the inflammatory mechanisms of idiopathic pulmonary fibrosis, and the results are in agreement with the hypothesis of a downregulation of the T-helper 1 immunological response in this disease.

Vestibular memory-contingent whole-body return: brave exocentered dancers.

A whole-body, self-driven return from passive rotation (90 degrees to 270 degrees ) test was executed by male and female subjects, and by professional female ballet dancers. To accomplish the return task, subjects were free to use the egocentered reference frame (inversion) or the exocentered one (completion). The results show that with inversion all kinetic parameters were reproduced, whereas the completion performance was highly variable. Although inversion was the default strategy, female subjects used more completions than male subjects, and female dancers still more, although not more accurately. The high variability noted in completion shows a deficiency in integrating vestibular signals for updating the egocentric representation of an external target during passive body rotations, even in dancers. Furthermore, with completions after 180 degrees stimuli, the results suggest that both ego- and exocentered reference frames cannot be used simultaneously.

Nasopharyngeal teratoma and diaphragmatic hernia: a non random association?

We describe a female affected by diaphragmatic hernia and nasopharyngeal teratoma. The case is compared with one already reported and possible diagnoses discussed. These cases appear to represent a new syndrome.

Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview.

In the last decade, the analysis of bronchial biopsies and lung parenchyma obtained from chronic obstructive pulmonary disease (COPD) patients compared with those from smokers with normal lung function and non-smokers has provided new insights on the role of the different inflammatory and structural cells, their signalling pathways and mediators, contributing to a better knowledge of the pathogenesis of COPD. This review summarizes and discusses the lung pathology of COPD patients with emphasis on inflammatory cell phenotypes that predominate in different clinical conditions. In bronchial biopsies, a cascade of events takes place during progression from mild-to-severe disease. T lymphocytes, particularly CD8+ cells and macrophages are the prevalent inflammatory cells in the lung of healthy smokers and patients with mild COPD, while total and activated neutrophils predominate in severe COPD. The number of CD4+, CD8+ cells and macrophages expressing nuclear factor-kappa B (NF-kappaB), STAT-4 and IFN-gamma proteins as well as endothelial adhesion molecule-1 in endothelium is increased in mild/moderate disease. In contrast, activated neutrophils (MPO+ cells) and increased nitrotyrosine immunoreactivity develops in severe COPD. In bronchial biopsies obtained during COPD exacerbations, some studies have shown an increased T cell and granulocyte infiltration. Regular treatment with high doses of inhaled glucocorticoids does not significantly change the number of inflammatory cells in bronchial biopsies from patients with moderate COPD. The profile in lung parenchyma is similar to bronchial biopsies. 'Healthy' smokers and mild/moderate diseased patients show increased T lymphocyte infiltration in the peripheral airways. Pulmonary emphysema is associated with a general increase of inflammatory cells in the alveolar septa. The molecular mechanisms driving the lymphocyte and neutrophilic prevalence in mild and severe disease, respectively, needs to be extensively studied. Up-regulation of pro-inflammatory transcription factors NF-kappaB and STAT-4 in mild, activated epithelial and endothelial cells in the more severe disease may contribute to this differential prevalence of infiltrating cells.

STAT4 activation in smokers and patients with chronic obstructive pulmonary disease.

Activation of the transcription factor signal transducer and activator of transcription (STAT)-4 is critical for the differentiation of T-helper 1 cells/type-1 cytotoxic T-cells and the production of interferon (IFN)-gamma. Expression of STAT4, phospho-STAT4, IFN-gamma and T-box expressed in T-cells (T-bet) proteins in bronchial biopsies and bronchoalveolar lavage (BAL)-derived lymphocytes, obtained from 12 smokers with mild/moderate chronic obstructive pulmonary disease (COPD) (forced expiratory volume in one second (FEV1) 59 +/- 16% predicted), 14 smokers with normal lung function (FEV1 106 +/- 12% pred) and 12 nonsmoking subjects (FEV1 111 +/- 14% pred), was examined by immunohistochemistry and immunocytochemistry. In bronchial biopsies of COPD patients, the number of submucosal phospho-STAT4+ cells was increased (240 (22-406) versus 125 (0-492) versus 29 (0-511) cells mm(-2)) when compared with both healthy smokers and control nonsmokers, respectively. In smokers, phospho-STAT4+ cells correlated with the degree of airflow obstruction and the number of IFN-gamma+ cells. Similar results were seen in BAL (2.8 (0.2-5.9) versus 1.03 (0.09-1.6) versus 0.69 (0-2.3) lymphocytes x mL(-1) x 10(3)). In all smokers who underwent lavage, phospho-STAT4+ lymphocytes correlated with airflow obstruction and the number of IFNgamma+ lymphocytes. T-bet expression was not altered in bronchial biopsies and BAL-derived lymphocytes between the three groups. In conclusion, this study suggests that stable mild/moderate chronic obstructive pulmonary disease is associated with an active T-helper 1 cell/type-1 cytotoxic T-cell inflammatory process involving activation of signal transducer and activator of transcription 4 and interferon-gamma production.

Effect of folic acid on homocysteine-induced trophoblast apoptosis.

In trophoblast cells exposed to homocysteine (Hcy) we observed cellular apoptosis and the inhibition of trophoblast functions. Because folate and Hcy, linked in the same metabolic pathway, are inversely related, we investigated the role of folic acid in reversing the Hcy effect in human placenta. In primary trophoblast cells we examined the cytosolic release of cytochrome c, both M30 and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and DNA laddering. Hcy (20 micromol/l) treatment resulted in cytochrome c release from mitochondria to the cytosol, and an increased number of M30-positive trophoblast cells and TUNEL positive nuclei. Furthermore, DNA cleavage in agarose gel and the determination of histone-associated DNA fragments have been investigated. Homocysteine induced DNA fragmentation and significantly reduced hCG secretion. The addition of folic acid (20 nmol/l) resulted in inhibition of the effects of Hcy on human trophoblast. These results suggest a protective role of folic acid in the prevention of trophoblast apoptosis linked to Hcy.

Multifactorial interactions involved in linear self-transport distance estimate: a place for time.

As the vestibular system is the only sensory organ whose primary function is self-motion detection, we examined the conditions under which the otoliths, which detect the linear acceleration of the head, could be used to estimate traveled distance. In order to isolate the contribution of the otoliths (with the somatosensory system) from contributions of the visual and motor systems subjects were transported in darkness. We initially hypothesized that self-transport with continuously varying linear velocity should facilitate distance computation by continuously stimulating the otoliths, and that active control of self-motion should also help subjects estimate the distance traveled. However, it was found that the distance covered during self-motion is actually better estimated when transport velocity is quasi-constant. Nevertheless, such estimates strongly depend upon velocity magnitude; subjects show an idiosyncratic preferred self-motion velocity for which distance measurements are most accurate. Furthermore, the active control of self-transport improves estimates of self-motion mainly because the subjects can then adopt a constant velocity, and more precisely their preferred one. It was finally found that subjects mentally count in order to assess their displacement length, and that time perception is indeed disturbed by varying self-motion velocity.

Isolated PACNS-like presentation of a systemic vasculitis complicating a myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are a series of haematological malignancies ranging from chronic refractory anaemia to leukaemia. There is increasing recognition of immunological abnormalities in patients with MDS, including few reports of cutaneous vasculitis; in no instance, a cerebral localization has been ascertained. Here, the case of a patient with MDS who presented exclusively with neurological signs that were considered indicative of a primary, isolated central nervous system vasculitis (PACNS) is reported. Although histological findings on brain tissue confirmed a small-vessel vasculitis, this had to be considered in the context of a systemic vasculitis. In fact, at autopsy, an involvement of skin, myocardium, lungs, liver, kidney and bone marrow was also found. An autoimmune vasculitis should be included in the differential diagnosis of acute-onset, isolated, cerebral symptoms complicating the course of MDS.

Lower respiratory tract infections in chronic obstructive pulmonary disease outpatients with tracheostomy and persistent colonization by P. aeruginosa.

Outpatients with tracheostomy can be managed with a low risk for severe airways infections despite colonization with pathogenic bacteria. No studies have been focused on chronic obstructive pulmonary disease (COPD), a condition known for recurrent exacerbations. The aim of our study was to verify whether at follow-up in tracheostomized COPD versus other disease outpatients, persistent P. aeruginosa colonization may influence the rate and treatment of lower respiratory tract infections (LRTI) or hospital admissions. Thirty-nine outpatients were considered: 24 were affected by COPD (age 66, 54-78 years, mean, range), 15 by restrictive lung disease (RLD) (57, 41-72 years). During an 18-month follow-up the number of LRTIs were recorded. Bacterial identifications were assessed at baseline and every month for 6 months in bronchial aspirates. The number of LRTI per patient was not significantly different between COPD [37, 1(0-6)] and RLD [18, 1(0-5)], [total, median (range)]. Persistent P. aeruginosa colonized 18 COPD (75%), 12 RLD patients (86%) and was not associated with an increased number of LRTI: 1(0-6) and 1(0-2), respectively. There were no differences in the number of hospital admissions: COPD 0(0-2), RLD 1(0-1), with a significant decrease versus before tracheostomy (P < 0.001). In conclusion, the rate of LRTI and hospital admissions in COPD outpatients with chronic tracheostomy was low, similar to non-COPD patients and independent of P. aeruginosa colonization.