Inflammatory and functional effects of increasing asthma treatment with formoterol or double dose budesonide

Adding a long-acting beta(2)-agonist to inhaled corticosteroids (ICS) for asthma treatment is better than increasing ICS dose in improving clinical status, although there is no consensus about the impact of this regimen on inflammation. In this double-blind, randomized, parallel group study, asthmatics with moderate to severe disease used budesonide (400 mcg/day) for 5 weeks (run-in period); then they were randomized to use budesonide (800 mcg/day - BUD group) or budesonide plus formoterol (400 mcg and 24 mcg/day, respectively - FORMO group) for 9 weeks (treatment period). Home PEF measurements, symptom daily reporting, spirometry, sputum induction (for differential cell counts and sputum cell cultures), and hypertonic saline bronchial challenge test were performed before and after treatments. TNF-alpha, IL-4 and eotaxin-2 levels in the sputum and cell culture supernatants were determined. Morning and night PEF values increased in the FORMO group during the treatment period (p < 0.01), from 435 +/- 162 to 489 +/- 169 and 428 +/- 160 to 496 +/- 173 L/min, respectively. The rate of exacerbations in the FORMO group was lower than in the BUD group (p < 0.05). Neutrophil counts in sputum increased in both groups (p < 0.05) and leukocyte viability after 48 h-culture increased in the FORMO group (p < 0.05). No other parameter changed significantly in either group. This study showed that adding formoterol to budesonide improved home PEF and provided protection from exacerbations, although increase of leukocyte viability in cell culture may be a matter of concern and needs further investigation. (C) 2008 Elsevier Ltd. All rights reserved.

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146(+) perivascular cells and fibroblasts

Objective. The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. Materials and Methods. We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. Results. Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. Conclusion. Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

The peripheral pro-nociceptive state induced by repetitive inflammatory stimuli involves continuous activation of protein kinase A and protein kinase C epsilon and its Na(v)1.8 sodium channel functional regulation in the primary sensory neuron

In the present study, the participation of the Na(v)1.8 sodium channel was investigated in the development of the peripheral pro-nociceptive state induced by daily intraplantar injections of PGE(2) in rats and its regulation in vivo by protein kinase A (PKA) and protein kinase C epsilon (PKC epsilon) as well. In the prostaglandin E(2) (PGE(2))-induced persistent hypernociception, the Na(v)1.8 mRNA in the dorsal root ganglia (DRG) was up-regulated. The local treatment with dipyrone abolished this persistent hypernociception but did not alter the Na(v)1.8 mRNA level in the DRG. Daily intrathecal administrations of antisense Na(v)1.8 decreased the Na(v)1.8 mRNA in the DRG and reduced ongoing persistent hypernociception. once the persistent hypernociception had been abolished by dipyrone, but not by Na(v)1.8 antisense treatment, a small dose of PGE(2) restored the hypernociceptive plateau. These data show that, after a period of recurring inflammatory stimuli, an intense and prolonged nociceptive response is elicited by a minimum inflammatory stimulus and that this pro-nociceptive state depends on Na(v)1.8 mRNA up-regulation in the DRG. in addition, during the persistent hypernociceptive state, the PKA and PKC epsilon expression and activity in the DRG are up-regulated and the administration of the PKA and PKC epsilon inhibitors reduce the hypernociception as well as the Na(v)1.8 mRNA level. In the present study, we demonstrated that the functional regulation of the Na(v)1.8 mRNA by PKA and PKC epsilon in the primary sensory neuron is important for the development of the peripheral pro-nociceptive state induced by repetitive inflammatory stimuli and for the maintenance of the behavioral persistent hypernociception. (C) 2008 Elsevier Inc. All rights reserved.

Functional characterization of the hypothalamic-pituitary-adrenal axis of the Wistar Audiogenic Rat (WAR) strain

The Wistar Audiogenic Rat (WAR) strain is a genetic model of sound-induced reflex epilepsy which was selected starting from audiogenic seizures susceptible Wistar rats. Wistar resistant rats were used as WAR`s control in this study. In the acute situation, audiogenic seizures (AS) in WARs mimic tonic-clonic seizures and, in the chronic protocol, mimic temporal lobe epilepsy. AS have been shown to evoke neuroendocrine responses; however, the hypothalamic-pituitary-adrenal activity in the WAR has not been established. The aim of this study was to evaluate the hypothalamic-pituitary-adrenal axis (HPA) responses to exogenous ACTH stimulation (8 ng/rat), fifteen minute restraint stress and circadian variation (8 am and 8 pm) under rest conditions in these animals through plasma measurements of ACTH and corticosterone concentrations. We also measured the body weight from birth to the 9th week of life and determined adrenal gland weight. We found that WARs are smaller than Wistar and presented a higher adrenal gland weight with a higher level of corticosterone release after intravenous ACTH injection. They also showed altered HPA axis circadian rhythms and responses to restraint stress. Our data indicate that, despite the lower body weight, WARs have increased adrenal gland weight associated with enhanced pituitary and adrenal responsiveness after HPA axis stimulation. Thus, we propose WARs as a model to study stress-epilepsy interactions and epilepsy-neuropsychiatry comorbidities. (C) 2011 Elsevier B.V. All rights reserved.

Methods for exploring the morpho-functional relations of the aortic depressor nerve in experimental diabetes

The present study investigated morpho-functional relations of the aortic depressor nerve (ADN) 5, 15 and 120 days after the onset of streptozotocin-induced diabetes in rats. Time control animals received vehicle. Under pentobarbital anesthesia, ADN activity was recorded simultaneously with arterial pressure. After the recordings, nerves were prepared for light microscopy study and morphometry. ADN function was accessed by means of pressure-nerve activity curve (fitted by sigmoidal regression) and cross-spectral analysis between mean arterial pressure (MAP) and ADN activity. The relation between morphological (myelinated fibers number and density, total myelin area, total fiber area and percentage of occupancy) and functional (gain, signal/noise relation, frequency) parameters were accessed by linear regression analysis and correlation coefficient calculations. Functional parameters obtained by means of the sigmoidal regression curve as well as by cross-spectral analysis were similar in diabetic and control rats. Morphometric parameters of the ADN were similar between groups 5 days after the onset of diabetes. Average myelin area and myelinated fiber area were significantly smaller on diabetic rats 15 and 120 days after the onset of diabetes, being the myelinated fiber and respective axons area and diameter also smaller on 120 days group. Nevertheless, G ratio (ratio between axon and fiber diameter) was nearly 0.6 and not different between groups or experimental times. No significant relationship between morphological and functional parameters was detected in all experimental groups. The present study suggests that ADN diabetic neuropathy was time-dependent, with damage to myelinated fibers to be the primary event, not evidenced by physiological methods. (C) 2010 Elsevier B.V. All rights reserved.

Association study of an epidermal growth factor gene functional polymorphism with the risk and prognosis of gliomas in Brazil

Epidermal growth factor (EGF) plays an important role in cancer. A functional single nucleotide polymorphism (SNP) in the 5`-untranslated region of the EGF gene (+61 A>G) may influence its expression and contribute to cancer predisposition and aggressiveness. Aiming to investigate the role of EGF +61 A>G in the susceptibility to glioma and its prognosis, we performed a case-control study with 165 patients and 200 healthy controls from Brazil. Comparisons of genotype distributions and allele frequencies did not reveal any significant differences between the groups. The mean overall survival was 9.2 months for A/A, 8.2 months for A/G, and 7.7 months for GIG. When survival curves were plotted we found that the +61G allele is associated with poor overall survival (p=0.023) but not with disease-free survival (p=0.527). Our data suggest that, although there is no association between the EGF +61 A>G genotype and glioma susceptibility, this SNP is associated with shorter overall survival of glioma patients in the Brazilian population. Nevertheless, future studies utilizing a larger series are essential for a definitive conclusion. (Int J Biol Markers 2009; 24: 277-81)

Altered gray matter morphometry and resting-state functional and structural connectivity in social anxiety disorder

In social anxiety disorder (SAD), impairments in limbic/paralimbic structures are associated with emotional dysregulation and inhibition of the medial prefrontal cortex (MPFq. Little is known, however, about alterations in limbic and frontal regions associated with the integrated morphometric, functional, and structural architecture of SAD. Whether altered gray matter volume is associated with altered functional and structural connectivity in SAD. Three techniques were used with 18 SAD patients and 18 healthy controls: voxel-based morphometry; resting-state functional connectivity analysis; and diffusion tensor imaging tractography. SAD patients exhibited significantly decreased gray matter volumes in the right posterior inferior temporal gyrus (ITG) and right parahippocampal/hippocampal gyrus (PHG/HIP). Gray matter volumes in these two regions negatively correlated with the fear factor of the Liebowitz Social Anxiety Scale. In addition, we found increased functional connectivity in SAD patients between the right posterior ITG and the left inferior occipital gyrus, and between the right PHF/HIP and left middle temporal gyms. SAD patients had increased right MPFC volume, along with enhanced structural connectivity in the genu of the corpus callosum. Reduced limbic/paralimbic volume, together with increased resting-state functional connectivity, suggests the existence of a compensatory mechanism in SAD. Increased MPFC volume, consonant with enhanced structural connectivity, suggests a long-time overgeneralization of structural connectivity and a role of this area in the mediation of clinical severity. Overall, our results may provide a valuable basis for future studies combining morphometric, functional and anatomical data in the search for a comprehensive understanding of the neural circuitry underlying SAD. (C) 2011 Elsevier B.V. All rights reserved.

A functional study on gentamicin-related cochleotoxicity in its conventional dose in newborns

The early identification of hearing impairment allows for an intervention still in the ""critical"" and ideal period of hearing and language stimulation. Pediatric ototoxicity is a very controversial topic. There have been variable percentages of ototoxicity cases in children with different aminoglycosides antibiotics. The main pediatric groups whom receive aminoglycosides are newborns with severe infections on the neonatal ICU. Aim: to check the functional aspect of the cochlear external hair cells and treatment regimens used to treat infections during the neonatal period. Study design: Experimental. Materials and Methods: we studied 26 albino guinea pigs, through distortion product otoacoustic emissions, before and after the use of gentamicin. Results: in all the assessments, the external hair cells functional status, studied by means of the distortion product otoacoustic emissions, proved preserved. Conclusion: In the present study, we did not notice changes in outer hair cell function in the albino guinea pigs treated with gentamicin in the doses of 4 mg/Kg/ day and 2.5 mg/Kg/day every 12 hours for 10 and 14 days.

Functional deficiencies of sulfite oxidase: Differential diagnoses in neonates presenting with intractable seizures and cystic encephalomalacia

Sulfite oxidase is a mitochondrial enzyme encoded by the SUOX gene and essential for the detoxification of sulfite which results mainly from the catabolism of sulfur-containing amino acids. Decreased activity of this enzyme can either be due to mutations in the SUOX gene or secondary to defects in the synthesis of its cofactor, the molybdenum cofactor. Defects in the synthesis of the molybdenum cofactor are caused by mutations in one of the genes MOCS1, MOCS2, MOCS3 and GEPH and result in combined deficiencies of the enzymes sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Although present in many ethnic groups, isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are rare inborn errors of metabolism, which makes awareness of key clinical and laboratory features of affected individuals crucial for early diagnosis. We report clinical, radiologic, biochemical and genetic data on a Brazilian and on a Turkish child with sulfite oxidase deficiency due to the isolated defect and impaired synthesis of the molybdenum cofactor, respectively. Both patients presented with early onset seizures and neurological deterioration. They showed no sulfite oxidase activity in fibroblasts and were homozygous for the mutations c.1136A>G in the SUOX gene and c.667insCGA in the MOCS1 gene, respectively. Widely available routine laboratory tests such as assessment of total homocysteine and uric acid are indicated in children with a clinical presentation resembling that of hypoxic ischemic encephalopathy and may help in obtaining a tentative diagnosis locally, which requires confirmation by specialized laboratories. (C) 2009 Published by Elsevier B.V.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D(2)) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for `in-situ` hybridisation study using (35)S-labeled oligonucleotide probe complementary to D(2) receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D(2) receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D(2) receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Functional and bioinformatics analyses reveal conservation of Cis-regulatory elements between sciaridae and Drosophilidae

The sciarid DNA puff C4 BhC4-1 gene is amplified and transcribed in salivary glands at the end of the larval stage. In transgenic Drosophila, the BhC4-1 promoter drives transcription in prepupal salivary glands and in the ring gland of late embryos. A bioinformatics analysis has identified 162 sequences similar to distinct regions of the BhC4-1 proximal promoter, which are predominantly located either in 5` or 3` regions or introns in the Drosophila melanogaster genome. A significant number of the identified sequences are found in the regulatory regions of Drosophila genes that are expressed in the salivary gland. Functional assays in Drosophila reveal that the BhC4-1 proximal promoter contains both a 129 bp (-186/-58) salivary gland enhancer and a 67 bp (-253/-187) ring gland enhancer that drive tissue specific patterns of developmentally regulated gene expression, irrespective of their orientation.

The Stability of the Blood Oxygenation Level-Dependent Functional MRI Response to Motor Tasks Is Altered in Patients With Chronic Ischemic Stroke

Background and Purpose-Functional MRI is a powerful tool to investigate recovery of brain function in patients with stroke. An inherent assumption in functional MRI data analysis is that the blood oxygenation level-dependent (BOLD) signal is stable over the course of the examination. In this study, we evaluated the validity of such assumption in patients with chronic stroke. Methods-Fifteen patients performed a simple motor task with repeated epochs using the paretic and the unaffected hand in separate runs. The corresponding BOLD signal time courses were extracted from the primary and supplementary motor areas of both hemispheres. Statistical maps were obtained by the conventional General Linear Model and by a parametric General Linear Model. Results-Stable BOLD amplitude was observed when the task was executed with the unaffected hand. Conversely, the BOLD signal amplitude in both primary and supplementary motor areas was progressively attenuated in every patient when the task was executed with the paretic hand. The conventional General Linear Model analysis failed to detect brain activation during movement of the paretic hand. However, the proposed parametric General Linear Model corrected the misdetection problem and showed robust activation in both primary and supplementary motor areas. Conclusions-The use of data analysis tools that are built on the premise of a stable BOLD signal may lead to misdetection of functional regions and underestimation of brain activity in patients with stroke. The present data urge the use of caution when relying on the BOLD response as a marker of brain reorganization in patients with stroke. (Stroke. 2010; 41:1921-1926.)

Relationship among eccentric hip and knee torques, symptom severity and functional capacity in females with patellofemoral pain syndrome

Objective: To determine the relationships between eccentric hip and knee torques, symptom severity and functional capacity in females with patellofemoral pain syndrome (PFPS). Design: Within-subject correlational study. Setting: University biomechanics laboratory. Participants: 10 females diagnosed with PFPS. Main outcome measures: Eccentric strength of the hip abductors and lateral rotators, and knee extensors were assessed using an isokinetic dynamometer. A 10-cm visual analog scale was used to determine usual knee pain in the last week. The Anterior Knee Pain Scale (AKPS) was used to determine the functional capacity of the patients. Results: The study found that the greater the eccentric knee extensor and hip lateral rotator torques, the higher the functional capacity of the patients (p = 0.02, r = 0.72; p = 0.02, r = 0.72). It was also shown that the greater hip lateral rotator torque, the less the usual pain reported in the last week (p = 0.004, r = -0.84). Despite the lack of statistical significance (p = 0.11), it was also found a modest negative relationship between the eccentric knee extensor torque and the usual pain reported in the last week (r = -0.56) that was considered clinically meaningful (d = 1.4). Conclusions: This study showed that eccentric knee extensor and hip lateral rotator torques were associated with functional capacity and pain level in females with PFPS. Further investigations should be carried out to verify the effects of an intervention program focused on the eccentric action of these muscles with respect to the symptoms in patients with PFPS. (C) 2011 Elsevier Ltd. All rights reserved.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), SmA, is a vaccine candidate against, S. mansoni and F hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine. (C) 2008 Elsevier B.V. All rights reserved.

Absence of functional TLR4 impairs response of macrophages after Candida albicans infection

Candida albicans is recognized by phagocytic cells through a set of recognition receptors patterns. Recently, we showed the importance of TLR2 in the regulation of neutrophil survival after C. albicans infection. In the present work, we analyzed the involvement of TLR4 in the recognition of C. albicans by neutrophils and macrophages. Our results show that the absence of functional TLR4 resulted in lower chemotaxis of neutrophils to the site of infection, lower levels of TNF-alpha, CXCL1 and nitric oxide, and dissemination and persistence of the pathogen in lymph nodes and spleen. In vitro, the phagocytic activity, nitric oxide production and myeloperoxidase activity, CXCL1, IL-1 beta production by neutrophils from TLR4-defective mice were not changed. In contrast, macrophages from TLR4-defective mice demonstrated lower phagocytosis and lower levels of CXCL1, IL-1 beta and TNF-alpha. Together, these data demonstrate that TLR4 signals are important for the recognition of C. albicans by macrophages and their absence allows persistence of the infection.