CD8+ T lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis

Objective

To study the prevalence of respiratory and atopic symptoms in (young) adults born prematurely, differences between those who did and did not develop Bronchopulmonary Disease (BPD) at neonatal age and differences in respiratory health between males and females.

Methods

Design: Prospective cohort study. Setting: Nation wide follow-up study, the Netherlands. Participants: 690 adults (19 year old) born with a gestational age below 32 completed weeks and/or with a birth weight less than 1500 g. Controls were Dutch participants of the European Community Respiratory Health Survey (ECRHS). Main outcome measures: Presence of wheeze, shortness of breath, asthma, hay fever and eczema using the ECRHS-questionnaire

Results

The prevalence of doctor-diagnosed asthma was significantly higher in the ex-preterms than in the general population, whereas eczema and hay fever were significant lower. Women reported more symptoms than men. Preterm women vs controls: asthma 13% vs 5% (p < 0.001); hay fever 8% vs 20% (p < 0.001); eczema 10% vs 42% (p < 0.001). Preterm men vs controls: asthma 9% vs 4% (p = 0.007); hay fever 8% vs 17% (p = 0.005); eczema 9% vs 31% (p < 0.001) Preterm women reported more wheeze and shortness of breath during exercise (sob) than controls: wheeze 30% vs 22% (p = 0.009); sob 27% vs 16% (p < 0.001); 19-year-old women with BPD reported a higher prevalence of doctor diagnosed asthma compared to controls (24% vs 5% p < 0.001) and shortness of breath during exercise (43% vs 16% p = 0.008). The prevalence of reported symptoms by men with BPD were comparable with the controls.

Conclusion

Our large follow-up study shows a higher prevalence of asthma, wheeze and shortness of breath in the prematurely born young adults. 19-year-old women reported more respiratory symptoms than men. Compared to the general population atopic diseases as hay fever and eczema were reported less often.     

Structural and functional characterization of the dimerization region of soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, alpha(1) beta(1), identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the beta(1) subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length alpha(1) in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204-408 mediates binding of beta(1) to alpha(1) The same region of beta(1)[204-408] was found to promote beta /beta(1) homodimerization. Fusion of [204 beta(1)-408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of beta(1)[204-408] with alpha(1) or beta(1) targeted the sGC subunits for proteasomal degradation, suggesting that beta(1)[204-408] forms structurally deficient complexes with alpha(1) and beta(1). Analysis of deletion constructs lacking portions of the beta(1) dimerization region identified two distinct segments contributing to alpha(1) binding, i.e. an N-terminal site covering positions 204-244 and a C-terminal site at 379-408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of beta(1) extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of beta(1) to the alpha(1) subunit of sGC.     

Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24h of septic onset, Interleukins (IL)-8, -1beta, -6, -10, and -12p70, tumor necrosis factor-alpha (TNF-alpha), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p<0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p<0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p<0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR=1.3, p=0.01 for CD64 and OR=1.26, p=0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

On the origin and evolution of thermophily: reconstruction of functional precambrian enzymes from ancestors of Bacillus

Thermophily is thought to be a primitive trait, characteristic of early forms of life on Earth, that has been gradually lost over evolutionary time. The genus Bacillus provides an ideal model for studying the evolution of thermophily as it is an ancient taxon and its contemporary species inhabit a range of thermal environments. The thermostability of reconstructed ancestral proteins has been used as a proxy for ancient thermal adaptation. The reconstruction of ancestral "enzymes" has the added advantages of demonstrable activity, which acts as an internal control for accurate inference, and providing insights into the evolution of enzymatic catalysis. Here, we report the reconstruction of the structurally complex core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, E. C. 1.1.1.85) from the last common ancestor (LCA) of Bacillus using both maximum likelihood (ML) and Bayesian inference. ML LeuB from the LCA of Bacillus shares only 76% sequence identity with its closest contemporary homolog, yet it is fully functional, thermophilic, and exhibits high values for k(cat), k(cat)/K(M), and ΔG(?) for unfolding. The Bayesian version of this enzyme is also thermophilic but exhibits anomalous catalytic kinetics. We have determined the 3D structure of the ML enzyme and found that it is more closely aligned with LeuB from deeply branching bacteria, such as Thermotoga maritima, than contemporary Bacillus species. To investigate the evolution of thermophily, three descendents of LeuB from the LCA of Bacillus were also reconstructed. They reveal a fluctuating trend in thermal evolution, with a temporal adaptation toward mesophily followed by a more recent return to thermophily. Structural analysis suggests that the determinants of thermophily in LeuB from the LCA of Bacillus and the most recent ancestor are distinct and that thermophily has arisen in this genus at least twice via independent evolutionary paths. Our results add significant fluctuations to the broad trend in thermal adaptation previously proposed and demonstrate that thermophily is not exclusively a primitive trait, as it can be readily gained as well as lost. Our findings also demonstrate that reconstruction of complex functional Precambrian enzymes is possible and can provide empirical access to the evolution of ancient phenotypes and metabolisms.     

Attitudes toward Infection Prophylaxis in Pediatric Oncology: A Qualitative Approach

Background

The risks and benefits of infection prophylaxis are uncertain in children with cancer and thus, preferences should be considered in decision making. The purpose of this report was to describe the attitudes of parents, children and healthcare professionals to infection prophylaxis in pediatric oncology.

Methods

The study was completed in three phases: 1) An initial qualitative pilot to identify the main attributes influencing the decision to use infection prophylaxis, which were then incorporated into a discrete choice experiment; 2) A think aloud during the discrete choice experiment in which preferences for infection prophylaxis were elicited quantitatively; and 3) In-depth follow up interviews. Interviews were recorded verbatim and analyzed using an iterative, thematic analysis. Final themes were selected using a consensus approach.

Results

A total of 35 parents, 22 children and 28 healthcare professionals participated. All three groups suggested that the most important factor influencing their decision making was the effect of prophylaxis on reducing the chance of death. Themes of importance to the three groups included antimicrobial resistance, side effects of medications, the financial impact of outpatient prophylaxis and the route and schedule of administration.

Conclusion

Effect of prophylaxis on risk of death was a key factor in decision making. Other identified factors were antimicrobial resistance, side effects of medication, financial impact and administration details. Better understanding of factors driving decision making for infection prophylaxis will help facilitate future implementation of prophylactic regiments.