Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Respiratory syncytial virus seasonality in Brazil: implications for the immunisation policy for at-risk populations

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalisation for respiratory diseases among children under 5 years old. The aim of this study was to analyse RSV seasonality in the five distinct regions of Brazil using time series analysis (wavelet and Fourier series) of the following indicators: monthly positivity of the immunofluorescence reaction for RSV identified by virologic surveillance system, and rate of hospitalisations per bronchiolitis and pneumonia due to RSV in children under 5 years old (codes CID-10 J12.1, J20.5, J21.0 and J21.9). A total of 12,501 samples with 11.6% positivity for RSV (95% confidence interval 11 - 12.2), varying between 7.1 and 21.4% in the five Brazilian regions, was analysed. A strong trend for annual cycles with a stable stationary pattern in the five regions was identified through wavelet analysis of the indicators. The timing of RSV activity by Fourier analysis was similar between the two indicators analysed and showed regional differences. This study reinforces the importance of adjusting the immunisation period for high risk population with the monoclonal antibody palivizumab taking into account regional differences in seasonality of RSV.

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Respiratory quotient evolution during normal pregnancy: what nutritional or clinical information can we get out of it?

Food intake increases to a varying extent during pregnancy to provide extra energy for the growing fetus. Measuring the respiratory quotient (RQ) during the course of pregnancy (by quantifying O2 consumption and CO2 production with indirect calorimetry) could be potentially useful since it gives an insight into the evolution of the proportion of carbohydrate vs. fat oxidized during pregnancy and thus allows recommendations on macronutrients for achieving a balanced (or slightly positive) substrate intake. A systematic search of the literature for papers reporting RQ changes during normal pregnancy identified 10 papers reporting original research. The existing evidence supports an increased RQ of varying magnitude in the third trimester of pregnancy, while the discrepant results reported for the first and second trimesters (i.e. no increase in RQ), explained by limited statistical power (small sample size) or fragmentary data, preclude safe conclusions about the evolution of RQ during early pregnancy. From a clinical point of view, measuring RQ during pregnancy requires not only sophisticated and costly indirect calorimeters but appears of limited value outside pure research projects, because of several confounding variables: (1) spontaneous changes in food intake and food composition during the course of pregnancy (which influence RQ); (2) inter-individual differences in weight gain and composition of tissue growth; (3) technical factors, notwithstanding the relatively small contribution of fetal metabolism per se (RQ close to 1.0) to overall metabolism of the pregnant mother.

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

Immediate Effects of Neurodynamic Sliding versus Muscle Stretching on Hamstring Flexibility in Subjects with Short Hamstring Syndrome.

Background. Hamstring injuries continue to affect active individuals and although inadequate muscle extensibility remains a commonly accepted factor, little is known about the most effective method to improve flexibility. Purpose. To determine if an isolated neurodynamic sciatic sliding technique would improve hamstring flexibility to a greater degree than stretching or a placebo intervention in asymptomatic subjects with short hamstring syndrome (SHS). Study Design. Randomized double-blinded controlled trial. Methods. One hundred and twenty subjects with SHS were randomized to 1 of 3 groups: neurodynamic sliding, hamstring stretching, and placebo control. Each subject's dominant leg was measured for straight leg raise (SLR) range of motion (ROM) before and after interventions. Data were analyzed with a 3 × 2 mixed model ANOVA followed by simple main effects analyses. Results. At the end of the study, more ROM was observed in the Neurodynamic and Stretching groups compared to the Control group and more ROM in the Neurodynamic group compared to Stretching group. Conclusion. Findings suggest that a neurodynamic sliding technique will increase hamstring flexibility to a greater degree than static hamstring stretching in healthy subjects with SHS. Clinical Relevance. The use of neurodynamic sliding techniques to improve hamstring flexibility in sports may lead to a decreased incidence in injuries; however, this needs to be formally tested.

Quality of life in patients with respiratory allergy is influenced by the causative allergen.

OBJECTIVE We aimed to analyze health-related quality of life (HRQOL) in adults with newly diagnosed respiratory allergy according to the sensitization profile for relevant aeroallergens in their usual area of residence. METHODS We performed a cross-sectional, epidemiological, observational, descriptive, multicenter study in allergy clinics in Spain. The sample comprised adults diagnosed with rhinitis, asthma, or both caused by significant allergens in their residential area (olive and/or grass pollen or house dust mite). Allergic rhinitis was classified according to the Allergic Rhinitis and its Impact on Asthma guidelines; asthma was classified according to the Guía Españiola para el Manejo del Asma (Spanish Guideline on the Management of Asthma). HRQOL was studied according to the ESPRINT-15 questionnaire and Mini Asthma Quality of Life Questionnaire. Control of asthma was measured using the Asthma Control Questionnaire 5. RESULTS We studied 1437 patients. Rhinitis was the most common respiratory disease. The HRQOL of rhinitis patients was lower in those sensitized to olive pollen only and in those with combined sensitization to olive and grass pollens. HRQOL associated with rhinitis was worse in patients diagnosed with both rhinitis and asthma than in patients diagnosed with rhinitis only. Asthma patients sensitized to olive pollen or olive and grass pollens had worse HRQOL. CONCLUSIONS In our study population, the HRQOL of patients with respiratory allergies varied with the allergen responsible for symptoms. In patients with rhinitis, the presence of asthma significantly worsened rhinitis-associated HRQOL.

Mitofusins 1/2 and ERRalpha expression are increased in human skeletal muscle after physical exercise

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of insulin resistance. Mitofusin (Mfn) proteins regulate the biogenesis and maintenance of the mitochondrial network, and when inactivated, cause a failure in the mitochondrial architecture and decreases in oxidative capacity and glucose oxidation. Exercise increases muscle mitochondrial content, size, oxidative capacity and aerobic glucose oxidation. To address if Mfn proteins are implicated in these exercise-induced responses, we measured Mfn1 and Mfn2 mRNA levels, pre-, post-, 2 and 24 h post-exercise. Additionally, we measured the expression levels of transcriptional regulators that control mitochondrial biogenesis and functions, including PGC-1alpha, NRF-1, NRF-2 and the recently implicated ERRalpha. We show that Mfn1, Mfn2, NRF-2 and COX IV mRNA were increased 24 h post-exercise, while PGC-1alpha and ERRalpha mRNA increased 2 h post-exercise. Finally, using in vitro cellular assays, we demonstrate that Mfn2 gene expression is driven by a PGC-1alpha programme dependent on ERRalpha. The PGC-1alpha/ERRalpha-mediated induction of Mfn2 suggests a role of these two factors in mitochondrial fusion. Our results provide evidence that PGC-1alpha not only mediates the increased expression of oxidative phosphorylation genes but also mediates alterations in mitochondrial architecture in response to aerobic exercise in humans

Cardio-pulmonary interaction in premature newborn with nasal continuous positive airways pressure (n-CPAP) respiratory assistance evaluated with echocardiography

Introduction: Nasal continuous positive airways pressure (n-CPAP) is an effective treatment in premature infants with respiratory distress. The cardio-pulmonary interactions secondary to n-CPAP are well studied in adults, but less well described in premature infants. We postulated that there could be important interactions with regard to the patent ductus arteriosus (PDA). Methods: Prospective study, approved by the local ethic committee. Premature infants less than 32 weeks gestation, _7 days-old, needing n-CPAP for respiratory distress, but without the need of additional oxygen were included in the study. Every patient had a first echocardiography with n-CPAP and then n-CPAP was retrieved. 3 hours later the echocardiography was repeated by the same investigator and then the patient replaced on n-CPAP. Results: 14 premature newborn were included, mean gestational age of 28 _ 2 weeks, mean weight 1.1 _ 0.3 Kg and height 39 _ 3 cm. Echocardiographic measurements are depicted in Table 1. Significant finding were observed between measurement on n- CPAP or without n-CPAP: on end diastolic left ventricular diameter (12.8 _ 1.6 mm vs. 13.5 _ 2 mm), on end systolic left ventricular diameter (8.4 _ 1.3 mm vs. 9.1 _ 1.5 mm), left atrium diameter (8.9 _ 2.2 mm vs. 10.4 _ 2.5 mm), maximal velocity on tricuspid valve (46 _ 10 cm/s vs. 51 _ 9 cm/s), calculated Qp (3.7 _ 0.8 L/min/m2 vs. 4.3 _ 0.8 L/min/m2). Only three patients have demonstrated a PDA during the study. Conclusion: Positive end expiratory pressure (Peep) has hemodynamic effects which are: reduction of systemic and pulmonary venous return as shown by the changes on tricuspid valve inflow,on the calculated Qp and finally on the diameter of the left atrium and left ventricle.We found in premature infants the same hemodynamic effects than those described in adults but with lower Peep values. This could be due to the particular elasticity and weakness of the thoracic wall of premature infants. Interestingly the flow through a PDA seems also to be diminished with Peep, but the number of patients is insufficient to conclude. Further investigation will be needed to better understand these interactions. Table 1. Echocardiographic measurement (mean (SD)). With n-CPAP Without n-CPAP p value RV ED diameter (mm) 6.3 (1.7) 6.04 (1.1) NS LV ED diameter (mm) 12.8 (1.6) 13.5 (2.0) _0.05 LV ES diameter (mm) 8.4 (1.3) 9.1 (1.5) _0.05 SF (%) 34 (5) 33 (6) NS Ao valve diameter (mm) 7.4 (1.3) 7.4 (1.2) NS LA diameter (mm) 8.9 (2.2) 10.4 (2.5) _0.05 Vmax Ao (cm/s) 70 (16) 71 (18) NS Vmax PV (cm/s) 69 (15) 72 (16) NS Vmax TV (cm/s) 46 (10) 51 (9) _0.05 Vmax MV (cm/s) 53 (17) 54 (18) NS Qp (L/min/m2) 3.7 (0.8) 4.3 (0.8) _0.05 Qs (L/min/m2) 4.0 (0.8) 4.0 (0.7) NS Qp/Qs 0.92 (0.14) 1.09 (0.23) _0.05 RV: right ventricle, LV: left ventricle, ED: end diastolic, ES: end systolic, SF: shortening fraction,Ao: aortic valve, LA: left atrium,Vmax: maximum Doppler Velocity, Qp: pulmonary output, Qs: systemic output, NS: non significant.

A comparative study of reactive hyperemia in human forearm skin and muscle

Résumé en français: L'hyperémie réactive dans la microcirculation musculature et cutanée de l'avant-bras permet d'évaluer l'atteinte vasculaire dans les maladie cardiovasculaires. Cette méthode permet d'obtenir un reflet de la progression de l'atteinte vasculaire, de traquer la progression de la maladie ainsi que le risque cardio-vasculaires. Elle est en étude également pour tester l'efficacité d'une intervention thérapeutique. L'hyperémie réactive est dépendante d'une dilatation post ischémique par diminution des résistances artériolaires. Au niveau des membres, l'ischémie peut-être débutée et interrompue très facilement par une manchette à pression gonflée au-dessus de la pression systolique suivie quelques minutes plus tard de son dégonflement. Les mesures de flux sanguin musculaire et cutané au niveau d'un membre sont facile à réaliser chez l'homme, tout particulièrement au niveau de l'avant-bras. Pour l'instant aucune étude utilisant cette approche ne spécifiait quel avant-bras était utilisé. Il est cependant concevable que la réponse varie selon que l'on teste le bras dominant ou non-dominant. Il parait donc important de clarifier ce point. Le premier but de l'étude consiste donc à investiguer une éventuelle différence entre le bras dominant et le bras non-dominant d'un sujet lors de tests de l'hyperémie réactive dans le muscle et la peau. Il est connu que l'hyperémie réactive au niveau musculaire peut-être diminuée par les médicaments antiinflammatoires non stéro~idiens (AINS), indiquant une implication partielle des métabolites de la cyclo-oxygénase. L'influence des AINS sur la réponse cutanée est moins clairement établie. Ainsi, le second but de cette étude est de comparer l'effet de l'inhibition de la cyclo-oxygénase sur l'hyperémie réactive musculaire et cutanée chez des sujets sains. Le collectif de patients consiste en 23 sujets masculins volontaires, en bonne santé, non fumeurs, de 18 à 30 ans. Aucuns antécédents médicaux ne sont connus et aucune médication n'est prise durant la période de l'étude. Tous . les sujets ont donné leur consentement par écrit. Le flux sanguin musculaire de l'avant-bras est mesuré au moyen d'une pléthysmographie par occlusion veineuse, et le flux cutané l'est par imagerie laser Doppler. Les expériences ont lieu entre 16 et 18 h dans une chambre calme à température constante (23-24°C) chez un sujet couché. Les participants n'ont pas consommé d'AINS durant la semaine précédente ni bu de café dans les 12 h précédant l'expérience. Les mesures sont effectuées en triplicat au niveau musculaire puis cutané ou inversement selon un ordre aléatoire. Suite à une occlusion artérielle l'étude du flux se fait sur 3 min et 5 min de récupération sont prises entre 2 mesures. L'expérience 1 consiste à tester un possible effet systématique de la latéralisation du bras dominant ou non sur la réponse à l'hyperémie réactive dans la peau et le muscle de l'avant-bras. 16 sujets sont étudiés à 2 reprises, espacées de 1 à 3 jours. A la première visite, l'hyperémie musculaire est étudiée dans un avant-bras, la réaction cutanée dans l'autre et inversement lors de la deuxième visite. Une précaution est observée afin de mesurer le flux sanguin cutané à la même distance du poignet dans les 2 avant-bras. L'expérience 2 est développée pour évaluer l'impact d'une inhibition des cyclo-oxygénases. Sept sujet sont considérés à 2 occasions espacées de 7 à 10 j. L'étude s'effectue uniquement au niveau de l'avant-bras dominant. Le site cutané au niveau du poignet est marqué lors de la première visite afin d'utiliser le même site de mesure lors de la seconde visite. Le sujet ingère 1,8 g d'Aspegic (équivalant à 1 g d'acide acétylsalilcylique) dissout dans 125 ml de jus d'orange ou le jus d'orange seul lors de l'autre visite selon un ordre randomisé. Les mesures sont débutées 2 h après la prise. Summary Reactive hyperemia (RH) in forearm muscle or skin microcirculation has been considered as a surrogate endpoint in clinical studies of cardiovascular disease. We evaluated two potential confounders that might limit such use of RH, namely laterality of measurement and intake of non-steroidal anti-inflammatory drugs (NSAIDS). Twenty-three young non-smoking healthy adults were enrolled. In Experiment 1 (n=16), the RH elicited by 3 min of ischemia was recorded in the muscle (strain gauge plethysmography, hand excluded) and skin (laser Doppler imaging) of both forearms. In Experiment 2 (n=7), RH was determined in the dominant forearm only, one hour following oral acetylsalicylic acid (1 g) or placebo. In Experiment 1, peak RH was identical in both forearms, and so were the corresponding durations of responses. RH lasted significantly less in muscle than in skin (p=0.003), a hitherto unrecognized fact. In the skin, acetylsalicylate reduced duration (43 vs 57.4 s for placebo, p=0.03), without affecting the peak response. In muscle, duration tended to decrease with acetylsalicylate (21.4 vs 26.0 s with placebo, p=0.06) and the peak increase in blood flow was blunted (27.2 vs 32.4 ml/min/100 ml tissue with placebo, p=0.003). We conclude that, when using RH as a surrogate endpoint in studies of cardiovascular disease, a confounding by laterality of measurement need not be feared, but NSAIDS may have an influence, although perhaps not on the peak response in the skin.

Kartagener syndrome: an uncommon cause of neonatal respiratory distress?

We report a newborn with respiratory distress and situs inversus totalis. The diagnosis of primary ciliary dyskinesia was confirmed by both ultrastructural and functional investigations. The immotile cilia syndrome was suspected because of respiratory distress, situs inversus, abnormal nasal discharge and hyperinflated chest X-ray. We suggest that ultrastructural and functional investigations of the respiratory mucosa should be done in any newborn with respiratory distress without explanation for the respiratory problems. Establishment of the correct diagnosis at an early stage may allow to improve the prognosis provided prophylactic physiotherapy, vaccinations, and aggressive antibiotic treatment of intercurrent respiratory infections are instituted. CONCLUSION Despite its rarity, primary ciliary dyskinesia should be considered in unexplained cases of neonatal distress.

A new paradigm of neuromuscular electrical stimulation for the quadriceps femoris muscle.

PURPOSE: Neuromuscular electrical stimulation (NMES) with large electrodes and multiple current pathways (m-NMES) has recently been proposed as a valid alternative to conventional NMES (c-NMES) for quadriceps muscle (re)training. The main aim of this study was to compare discomfort, evoked force and fatigue between m-NMES and c-NMES of the quadriceps femoris muscle in healthy subjects. METHODS: Ten healthy subjects completed two experimental sessions (c-NMES and m-NMES), that were randomly presented in a cross-over design. Maximal electrically evoked force at pain threshold, self-reported discomfort at different levels of evoked force, and fatigue-induced force declines during and following a series of 20 NMES contractions were compared between c-NMES and m-NMES. RESULTS: m-NMES resulted in greater evoked force (P < 0.05) and lower discomfort in comparison to c-NMES (P < 0.05-0.001), but fatigue time course and magnitude did not differ between the two conditions. CONCLUSIONS: The use of quadriceps m-NMES appears legitimate for (re)training purposes because it generated stronger contractions and was less discomfortable than c-NMES (due to multiple current pathways and/or lower current density with larger electrodes).

Neonatal treatment with recombinant ectodysplasin prevents respiratory disease in dogs with X-linked ectodermal dysplasia.

Patients with defective ectodysplasin A (EDA) have X-linked hypohidrotic ectodermal dysplasia (XLHED; OMIM#305100), a condition comprising hypotrichosis, inability to sweat, abnormal teeth, and frequent pulmonary infections. The XLHED dogs show the same clinical signs as humans with the disorder, including frequent respiratory infections that can be fatal. The respiratory disease in humans and dogs is thought to be due to the absence of tracheal and bronchial glands which are a vital part of the mucociliary clearance mechanism. In our XLHED model, the genetically missing EDA was replaced by postnatal intravenous administration of recombinant EDA resulting in long-term, durable corrective effect on adult, permanent dentition. After treatment with EDA, significant correction of the missing tracheal and bronchial glands was achieved in those dogs that received higher doses of EDA. Moreover, successful treatment resulted in the presence of esophageal glands, improved mucociliary clearance, and the absence of respiratory infection. These results demonstrate that a short-term treatment at a neonatal age with a recombinant protein can reverse a developmental disease and result in vastly improved quality of life.