Northern Ireland Chest Heart & Stroke - Research Grants for 2010 in Respiratory and Cardiovascular Disease

To download an Application Form, Guidance Notes and other information, please visit the website www.nichsa.com or contact Caoimhe Devlin, Research Assistant by email: cdevlin@nichsa.com or phone: 02890 266714 / 02890 320184 ext 248.

Respiratory syncytial virus infections during an epidemic period in Salvador, Brazil: viral antigenic group analysis and description of clinical and epidemiological aspects

Acute respiratory infections (ARI) caused by respiratory syncytial virus (RSV) were studied in 482 children from Salvador, BA, Brazil, over a period of 12 months. The epidemic period of RSV infections in Salvador occurred from February (summer) to August (winter), with peaks in May, June, and July. The grouping characteristics of 84 RSV present in nasopharyngeal secretions of children seen at a reference university hospital were analyzed. RSV represented 17.4% of all cases and 54.5% of the positive samples. Sixty-four RSV strains were assigned to group A and 14 to group B. Both groups circulated in the five months of the epidemic period studied. Infections by both groups of RSV were more frequent in children up to one year of age. The incidence of RSV ARI was slightly more frequent in males, although group B had more infected females.

Respiratory motion artifact suppression in diffusion-weighted MR imaging of the spine.

Diffusion-weighted spin-echo imaging of the spine has been successfully implemented for differentiation of benign fracture edema and tumor infiltration of the vertebral body. Nevertheless, this technique still suffers from insufficient image quality in numerous patients due to motion artifacts. The aim of this study was to investigate the impact of variable respiratory motion artifact suppression techniques on image quality in diffusion-weighted spin-echo imaging of the spine. In addition to phase-encoding reordering, a newly implemented right hemi-diaphragmaitc navigator for respiratory gating was used. Subjective and objective image quality parameters were compared. Respiratory motion artifact suppression has a major impact on image quality in diffusion-weighted imaging of the spine. Phase-encoding reordering does not enhance image quality while right hemi-diaphragmatic respiratory navigator gating significantly improves image quality at the cost of data acquisition time. Navigator gating should be used if standard spin-echo diffusion-weighted imaging demonstrates insufficient image quality.

Respiratory viruses in the pediatric intensive care unit: prevalence and clinical aspects

A survey was conducted in two pediatric intensive care units in hospitals in Porto Alegre, Brazil, in order to monitor the main respiratory viruses present in bronchiolitis and/or pneumonia and their involvement in the severity of viral respiratory infections. Viral respiratory infection prevalence was 38.7%. In bronchiolitis, respiratory syncytial virus (RSV) was detected in 36% of the cases. In pneumonia, the prevalence rates were similar for adenovirus (10.3%) and RSV (7.7%). There was a difference among the viruses detected in terms of frequency of clinical findings indicating greater severity. Frequency of crackles in patients with RSV (47.3%) showed a borderline significance (p = 0.055, Fisher's exact test) as compared to those with adenovirus (87.5%). The overall case fatality rate in this study was 2.7%, and adenovirus showed a significantly higher case fatality rate (25%) than RSV (2.8%) (p = 0.005). Injected antibiotics were used in 49% of the children with RSV and 60% of those with adenovirus. Adenovirus was not detected in any of the 33 children submitted to oxygen therapy.

Clinical characteristics and outcome of patients admitted to a medico- surgical ICU requiring non invasive ventilation (NIV) for hypercapnic respiratory failure

Introduction Because it decreases intubation rate and mortality, NIV has become first-line treatment in case of hypercapnic acute respiratory failure (HARF). Whether this approach is equally successful for all categories of HARF patients is however debated. We assessed if any clinical characteristics of HARF patients were associated with NIV intensity, success, and outcome, in order to identify prognostic factors. Methods Retrospective analysis of the clinical database (clinical information system and MDSi) of patients consecutively admitted to our medico-surgical ICU, presenting with HARF (defined as PaCO2 > 50 mmHg), and receiving NIV between May 2008 and December 2010. Demographic data, medical diagnoses (including documented chronic lung disease), reason for ICU hospitalization, recent surgical interventions, SAPS II and McCabe scores were extracted from the database. Total duration of NIV and the need for tracheal intubation during the 5 days following the first hypercapnia documentation, as well as ICU, hospital and one year mortality were recorded. Results are reported as median [IQR]. Comparisons were carried out with Chi2 or Kruskal-Wallis tests, p<0.05 (*). Results Two hundred and twenty patients were included. NIV successful patients received 16 [9-31] hours of NIV for up to 5 days. Fifty patients (22.7%) were intubated 11 [2-34] hours after HARF occurence, after having receiving 10 [5-21] hours of NIV. Intubation was correlated with increased ICU (18% vs. 6%, p<0.05) and hospital (42% vs. 31%, p>0.05) mortality. SAPS II score was related to increasing ICU (51 [29-74] vs. 23 [12-41]%, p<0.05), hospital (37% [20-59] vs 20% [12-37], p<0.05) and one year mortality (35% vs 20%, p<0.05). Surgical patients were less frequent among hospital fatalities (28.8% vs. 46.3%, p<0.05, RR 0.8 [0-6-0.9]). Nineteen patients (8.6%) died in the ICU, 73 (33.2%) during their hospital stay and 108 (49.1%) were dead one year after HARF. Conclusion The practice to start NIV in all suitable patients suffering from HARF is appropriate. NIV can safely and appropriately be used in patients suffering from HARF from an origin different from COPD exacerbation. Beside usual predictors of severity such as severity score (SAPS II) appear to be associated with increased mortality. Although ICU mortality was low in our patients, hospital and one year mortality were substantial. Surgical patients, although undergoing a similar ICU course, had a better hospital and one year outcome.

Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985-2001 - A review

The variability of the G glycoprotein from human respiratory syncytial viruses (HRSV) (groups A and B) isolated during 17 consecutive epidemics in Montevideo, Uruguay have been analyzed. Several annual epidemics were studied, where strains from groups A and B circulated together throughout the epidemics with predominance of one of them. Usually, group A predominates, but in some epidemics group B is more frequently detected. To analyse the antigenic diversity of the strains, extracts of cells infected with different viruses of group A were tested with a panel of anti-G monoclonal antibodies (MAbs). The genetic variability of both groups was analyzed by sequencing the C-terminal third of the G protein gene. The sequences obtained together with previously published sequences were used to perform phylogenetic analyses. The data from Uruguayan isolates, together with those from the rest of the world provide information regarding worldwide strain circulation. Phylogenetic analyses of HRSV from groups A and B show a model of evolution analogous to the one proposed for influenza B viruses providing information that would be beneficial for future immunization programs and to design safe vaccines.

Role of oxidative stress in plasticity of Dendritic cells

Dendritic cells (DCs) are antigen presenting cells with an unique ability to induce primary immune responses. Different DCs subsets with an intrinsic capacity to polarise Tcells have been described: myeloid (Th1) and lymphoid (Th2). Plasticity is defined as DCs capacity to polarise T cells independent of the DCs origin. We investigated the potential role played by oxidants such as superoxide anion (·O2-), in the plasticity of DCs, measured by the induction of a specific DCs subset, cytokine release and antigen presentation. Furthermore, we are interested in the amplification of immune response analysed by the exosomes production after oxidative stress and LPS stimulation. Recently, we have demonstrated that exposure of cells to superoxide anions resulted in the activation of DC2 profile. To analyse the role of oxidative stress in DCs subsets, we used BDCA-1 and BDCA-2 antibodies, which identify myeloid and plasmacytoid DCs respectively. Freshly isolated monocytes have shown to be BDCA-1-, but BDCA-2+ populations. During 6 days culture up-regulation of BDCA-1, but a down-regulation of BDCA-2 were observed, giving a clear myeloid population. When DC were stimulated with superoxide anions or LPS, we have observed that both down regulate the expression of BDCA-1 when compared to immature DC. Antigen presentation was markedly altered according to the periodicity used, and antigens and oxidants exposures. Using DCs trapped in collagen "matrix" after LPS activation we were able to quantify DCs-exosomes (small membrane vesicles ~50-100 nm in diameter) by reconstruction pictures in three dimensions. Using double vital staining we have found that exosomes from activated DCs can fuse with the membrane of resting DCs. Understanding the capacity of DCs to integrate external signals we will be able to unravel and control Tcells-polarisation triggering a specific immune response or tolerance. We will be able also to understand the amplification role of DCs-exosomes in remote not yet activated DCs.

Frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of Mexico City

A locality in the district of Tlalpan, Mexico City, was selected in order to identify the viral agents in children younger than 5 years of age with acute respiratory infection (ARI). A total of 300 children were randomly selected and were included in this study for a period of 13 months. During this period nasopharyngeal exudates were collected for the isolation of viral agents. Monoclonal fluorescent antibodies were used for viral identification after cell culture. Viral infection was detected in 65% of the specimens. The respiratory syncytial virus (RSV) was the most common virus agent detected. Children required an average of two consultations during the study period. Two high incidence peaks were observed, one during the summer and the other during winter; the most frequent viruses during these seasons were influenza A and RSV, respectively. The largest number of viruses was isolated in the group of children between 1 and 2 years of age and in the group between 4 and 5 years of age. This study demonstrated the presence of ARI and of different viruses in a period of 13 months, as well as the most frequent viruses in children younger than 5 years of age from a community of Mexico City.

Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlândia, MG, Brazil

The main viruses involved in acute respiratory diseases among children are: respiratory syncytial virus (RSV), influenzavirus (FLU), parainfluenzavirus (PIV), adenovirus (AdV), human rhinovirus (HRV), and the human metapneumovirus (hMPV). The purpose of the present study was to identify respiratory viruses that affected children younger than five years old in Uberlândia, Midwestern Brazil. Nasopharyngeal aspirates from 379 children attended at Hospital de Clínicas (HC/UFU), from 2001 to 2004, with acute respiratory disease, were collected and tested by immunofluorescence assay (IFA) to detect RSV, FLU A and B, PIV 1, 2, and 3 and AdV, and RT-PCR to detect HRV. RSV was detected in 26.4% (100/379) of samples, FLU A and B in 9.5% (36/379), PIV 1, 2 and 3 in 6.3% (24/379) and AdV in 3.7% (14/379). HRV were detected in 29.6% (112/379) of the negative and indeterminate samples tested by IFI. RSV, particularly among children less than six months of life, and HRV cases showed highest incidence. Negative samples by both IFA and RT-PCR might reflect the presence of other pathogens, such as hMPV, coronavirus, and bacteria. Laboratorial diagnosis constituted an essential instrument to determine the incidence of the most common viruses in respiratory infections among children in this region.

Isolation of respiratory syncytial virus from nasopharyngeal aspirates stored at 20ºC from one to fifteen months after collection

Cell culture isolation is used for recovering respiratory syncytial virus (RSV) from respiratory specimens. As RSV is a thermolabile virus, specimens destined for inoculation into cell culture require special transport, handling, and storage. The isolation rate of RSV from nasopharyngeal aspirates (NPA) stored at 20ºC for one to 15 months after collection was investigated. A total of 126 samples considered positive for RSV by indirect fluorescence-antibody were tested by virus isolation in HEp-2 cell culture. RSV was isolated from 47/126 specimens (37.3%). These results show that RSV may be recovered from NPA stored at 20ºC by cell culture.

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.

The European Respiratory Society spirometry tent: a unique form of screening for airway obstruction.

In order to raise public awareness of the importance of early detection of airway obstruction and to enable many people who had not been tested previously to have their lung function measured, the European Lung Foundation and the European Respiratory Society (ERS) organised a spirometry testing tent during the annual ERS Congresses in 2004-2009. Spirometry was performed during the ERS Congresses in volunteers; all participants answered a simple, brief questionnaire on their descriptive characteristics, smoking and asthma. Portable spirometers were freely provided by the manufacturer. Nurses and doctors from pulmonary departments of local hospitals/universities gave their service for free. Lower limit of normal (LLN) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria for diagnosing and grading airway obstruction were used. Of 12,448 participants in six congress cities, 10,395 (83.5%) performed acceptable spirometry (mean age 51.0 ± 18.4 yrs; 25.5% smokers; 5.5% asthmatic). Airway obstruction was present in 12.4% of investigated subjects according to LLN criteria and 20.3% according to GOLD criteria. Through multinomial logistic regression analysis, age, smoking habits and asthma were significant risk factors for airway obstruction. Relative risk ratio and 95% confidence interval for LLN stage I, for example, was 2.9 (2.0-4.1) for the youngest age (≤ 19 yrs), 1.9 (1.2-3.0) for the oldest age (≥ 80 yrs), 2.4 (2.0-2.9) for current smokers and 2.8 (2.2-3.6) for reported asthma diagnosis. In addition to being a useful advocacy tool, the spirometry tent represents an unusual occasion for early detection of airway obstruction in large numbers of city residents with an important public health perspective.

Appropriateness of respiratory care: development of evidence-based guidelines in an acute care hospital

Background: Respiratory care is universally recognised as useful, but its indications and practice vary markedly. In order to improve appropriateness of respiratory care in our hospital, we developed evidence-based local guidelines in a collaborative effort involving physiotherapists, physicians, and health services researchers. Methods: Recommendations were developed using the standardised RAND appropriateness method. A literature search was performed for the period between 1995 and 2008 based on terms associated with guidelines and with respiratory care. Publications were assessed according to the Oxford classification of quality of evidence. A working group prepared proposals for recommendations which were then independently rated by a multidisciplinary expert panel. All recommendations were then discussed in common and indications for procedures were rated confidentially a second time by the experts. Each indication for respiratory care was classified as appropriate, uncertain, or inappropriate, based on the panel median rating and the degree of intra-panel agreement. Results: Recommendations were formulated for the following procedures: non-invasive ventilation, continuous positive airway pressure, intermittent positive pressure breathing, intrapulmonary percussive ventilation, mechanical insufflation-exsufflation, incentive spirometry, positive expiratory pressure, nasotracheal suctioning, noninstrumental airway clearance techniques. Each recommendation referred to a particular medical condition, and was assigned to a hierarchical category based on the quality of evidence from literature supporting the recommendation and on the consensus of experts. Conclusion: Despite a marked heterogeneity of scientific evidence, the method used allowed us to develop commonly agreed local guidelines for respiratory care. In addition, this work fostered a closer relationship between physiotherapists and physicians in our institution.

Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

From January to December 1998, nasopharyngeal aspirates were obtained from 482 children with acute respiratory infections attended in emergence department and wards of a teaching hospital in the city of Salvador, Brazil. The samples were tested for the presence of adenovirus by isolation in tissue culture and indirect immunofluorescence assay. Eleven adenoviruses were detected by both methods in the same clinical samples. Infections by adenovirus were observed during seven months of the year without association with rainy season. Genome analysis was performed on these 11 isolates. Species C was represented by serotypes 1, 2 and 5. Within species B, only serotype 7 (Ad7) was detected. Two genomic variants of Ad1, two variants of Ad2, one of Ad5, and one of Ad7 (7h) were identified. This is the first study of molecular epidemiology of adenovirus associated to acute respiratory infections in children living in Northeast Brazil, and contributes to a better understanding of adenovirus infections in the country.

Endoglycan mediates malignant leukocyte rolling on e-selectin and signals through SYK and the MAPK pathway

Selectins play a key role regulating leukocyte migration into tissues by mediating leukocyte tethering (capture) and rolling on inflamed endothelium and/or on adherent leukocytes or platelets. During leukocyte rolling, endothelial E- or P-selectin bind to glycoprotein ligands carrying sialyl Lewis χ (sLex) determinant. P-selectin glycoprotein ligand-1 (PSGL-1) is a common ligand for L-, P- and E-selectin, which sequentially cooperates with CD44 and E- selectin ligand-1 (ESL-1) to roll on E-selectin. During rolling on endothelial selectins, PSGL-1 and CD44 signal through Src family kinases and Syk, leading to αι_β2 integrin partial activation and slow rolling on intercellular adhesion molecule-1 (ICAM-1). Leukocyte exposure to chemokines then leads to firm adhesion. Little information is available on ligands that mediate malignant leukocyte rolling on E- selectin. We defined these ligands on U937 monoblasts by immunoadsorbtion and immunoblotting using mAb raised against CD43, CD44, PSGL-1, sLex/CLA determinants and E-selectin/IgM chimera. Immunoblotting and blot rolling assays demonstrated that PSGL-1, CD43, CD44 and a -125 kDa sLex/CLA positive ligand contribute to support E-seiectin- dependent rolling. This -125 kDa ligand is endoglycan, a member of the CD34 family of sialomucins. Endoglycan was frequently detected by flow cytometry on primary leukemia, lymphoma and multiple myeloma ceils (in -50% of cases). Endoglycan, immunopurified from U937 cells, as well as endoglycan/IgG chimera efficiently supported E-selectin dependent rolling. Membrane fractionation on sucrose gradient demonstrated that endoglycan is expressed in lipid rafts. We tested the hypothesis that it signals, like PSGL-1 and CD44, through Src kinases and the MAPK pathway. Indeed, endoglycan engagement induced Syk and ERK phosphorylation in a iipid raft-dependent manner. Syk activation was dependent on Src kinase activity. Downstream of Syk, endoglycan activated PI3K and Akt as well as Bruton's tyrosine kinase and p38 MAPK. Thus, endoglycan is a ligand for endothelial selectins which may contribute to regulate leukemia, lymphoma and multiple myeloma cell trafficking and interactions with bone marrow microenvironment. - Les sélectines contrôlent la migration tissulaire des leucocytes en assurant leur capture et leur roulement sur l'endothélium vasculaire enflammé et/ou sur des plaquettes ou des leucocytes adhérant à la paroi vasculaire. Lors du roulement leucocytaire, les sélectines endothéliales (E- et P-sélectine) se lient à des ligands porteurs du saccharide sialyl Lewis χ (sLex). PSGL-1 est un ligand commun des sélectines qui coopère avec CD44 et ESL-1 pour permettre la capture et le roulement des neutrophiles. Lorsque PSGL-1 et CD44 se lient aux sélectines endothéliales, elles induisent la phosphorylation des kinases Src et de Syk conduisant à l'activation partielle de l'intégrine aLp2 et au ralentissement des leucocytes sur les sélectines et ICAM-1. Les chimiokines induisent ensuite l'adhésion ferme des leucocytes. Les ligands des sélectines qui assurent le roulement, sur la E-sélectine, des cellules issues d'hémopathies malignes sont peu connus. Nous avons caractérisé ces ligands en les purifiant avec des anticorps dirigés contre CD43, CD44, PSGL-1, sLex/CLA et en utilisant la chimère E-sélectine/IgM. Des tests d'adhésion ont montré que PSGL-1, CD43, CD44 et une glycoprotéine de ~125 kDa soutiennent les interactions cellulaires dépendant de la E- sélectine. Le ligand de -125 kDa a été identifié comme étant l'endoglycan. Il a été détecté, par cytométrie de flux, sur les cellules leucémiques, les cellules de lymphomes ou de myélome multiple, dans ~50% des cas analysés. Sa forme membranaire, immunopurifiée, ou recombinante (endoglycan/lgG) soutient les interactions cellulaires dépendant de la E- sélectine. Nous avons montré qu'il réside dans les rafts lipidiques membranaires puis avons testé l'hypothèse que l'endoglycan, comme PSGL-1 et CD44, induit une signalisation via les kinases de type Src et la voie des MAPK. Nous avons pu observer que son engagement induit la phosphorylation de Syk et de ERK pour autant que la structure des rafts soit préservée. En aval de Syk, l'endoglycan active la PI3K, Akt, Btk et la MAPK p38. Ces résultats montrent que l'endoglycan est un ligand des sélectines endothéliales qui pourrait participer au contrôle du trafic et des interactions des cellules leucémiques, de lymphomes ou de myélomes multiples avec leur microenvironnement. - Le sang est un élément clé du fonctionnement de notre corps. La circulation sanguine permet la communication et le transfert de molécules et cellules entre divers organes. Lors d'une inflammation aiguë due à une réaction allergique, une infection ou une blessure, on observe un oedème local accompagné de rougeur, de chaleur et souvent de douleurs. Au sein des tissus enflammés, on observe des globules blancs (leucocytes) et diverses molécules inflammatoires qui attirent les leucocytes dans les tissus lésés (chimiokines). Le sang est composé de globules rouges, de plaquettes et de leucocytes spécialisés dans les défenses immunes. Pour atteindre le site d'inflammation, les leucocytes doivent quitter la circulation sanguine. Ils utilisent pour cela des molécules d'adhésion présentes à leur surface qui se lient à d'autres molécules d'adhésion de la paroi sanguine. Leurs interactions permettent aux leucocytes de rouler à la surface du vaisseau sanguin. Lorsqu'ils roulent au voisinage d'un site d'inflammation, les leucocytes sont exposés à des chimiokines qui induisent leur arrêt et les dirigent dans les tissus enflammés. Ce processus physiologique est aussi impliqué dans des pathologies telles que l'infarctus, l'artériosclérose ou la thrombose. Il peut être détourné à des fins moins louables par des cellules cancéreuses pour permettre leur dissémination (métastatisation). Dans ce travail de thèse, nous avons caractérisé une molécule d'adhésion qui soutient l'adhésion des leucocytes aux sélectines endothéliales: l'endoglycan. Nous avons observé que cette molécule d'adhésion est fréquemment exprimée par les cellules malignes de nombreuses maladies du sang comme les leucémies, les lymphomes et le myélome multiple. Nous avons également pu montrer que l'endoglycan envoie des signaux à l'intérieur des cellules malignes lorsqu'elles se lient aux sélectines endothéliales. Ces signaux pourraient jouer un rôle déterminant dans la régulation des interactions des cellules malignes avec leur microenvironnement. Elles pourraient peut-être aussi favoriser leur survie et leur prolifération.