Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

<p>Abstract</p> <p>Background</p> <p>Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases.</p> <p>Methods</p> <p>sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. <it>In vitro </it>studies were performed to investigate which factors regulate sCD14 release and mCD14 expression.</p> <p>Results</p> <p>sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. <it>In vitro</it>, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition.</p> <p>Conclusions</p> <p>This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.</p

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

<p>Abstract</p> <p>Background</p> <p>Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases.</p> <p>Methods</p> <p>sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. <it>In vitro </it>studies were performed to investigate which factors regulate sCD14 release and mCD14 expression.</p> <p>Results</p> <p>sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. <it>In vitro</it>, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition.</p> <p>Conclusions</p> <p>This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.</p

Automated Analysis of Chest Radiographs for Cystic Fibrosis Scoring

We present a framework to analyze chest radiographs for cystic fibro-sis using machine learning methods. We compare the representational power of deep learning features with traditional texture features. Specifically, we respec-tively employ VGG-16 based deep learning features, Tamura and Gabor filter based textural features to represent the cystic fibrosis images. We demonstrate that VGG-16 features perform best, with a maximum agreement of 82%. In ad-dition, due to limited dimensionality, Tamura features for unsegmented images achieve no more than 50% agreement; however, after segmentation, the accuracy of Tamura can reach 78%. In combination with using the deep learning features, we also compare back propagation neural network and sparse coding classifiers to the typical SVM classifier with polynomial kernel function. The result shows that neural network and sparse coding classifiers outperform SVM in most cases. Only with insufficient training samples does SVM demonstrate higher accuracy

A new scoring system in Cystic Fibrosis: statistical tools for database analysis – a preliminary report

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis is the most common fatal genetic disorder in the Caucasian population. Scoring systems for assessment of Cystic fibrosis disease severity have been used for almost 50 years, without being adapted to the milder phenotype of the disease in the 21^stcentury. The aim of this current project is to develop a new scoring system using a database and employing various statistical tools. This study protocol reports the development of the statistical tools in order to create such a scoring system.</p> <p>Methods</p> <p>The evaluation is based on the Cystic Fibrosis database from the cohort at the Royal Children's Hospital in Melbourne. Initially, unsupervised clustering of the all data records was performed using a range of clustering algorithms. In particular incremental clustering algorithms were used. The clusters obtained were characterised using rules from decision trees and the results examined by clinicians. In order to obtain a clearer definition of classes expert opinion of each individual's clinical severity was sought. After data preparation including expert-opinion of an individual's clinical severity on a 3 point-scale (mild, moderate and severe disease), two multivariate techniques were used throughout the analysis to establish a method that would have a better success in feature selection and model derivation: 'Canonical Analysis of Principal Coordinates' and 'Linear Discriminant Analysis'. A 3-step procedure was performed with (1) selection of features, (2) extracting 5 severity classes out of a 3 severity class as defined per expert-opinion and (3) establishment of calibration datasets.</p> <p>Results</p> <p>(1) Feature selection: CAP has a more effective "modelling" focus than DA.</p> <p>(2) Extraction of 5 severity classes: after variables were identified as important in discriminating contiguous CF severity groups on the 3-point scale as mild/moderate and moderate/severe, Discriminant Function (DF) was used to determine the new groups mild, intermediate moderate, moderate, intermediate severe and severe disease. (3) Generated confusion tables showed a misclassification rate of 19.1% for males and 16.5% for females, with a majority of misallocations into adjacent severity classes particularly for males.</p> <p>Conclusion</p> <p>Our preliminary data show that using CAP for detection of selection features and Linear DA to derive the actual model in a CF database might be helpful in developing a scoring system. However, there are several limitations, particularly more data entry points are needed to finalize a score and the statistical tools have further to be refined and validated, with re-running the statistical methods in the larger dataset.</p

SBDS Expression and Localization at the Mitotic Spindle in Human Myeloid Progenitors

BACKGROUND: Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle. CONCLUSIONS/SIGNIFICANCE: SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34(+) hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained

Bone and body composition analyzed by Dual-energy X-ray Absorptiometry (DXA) in clinical and nutritional evaluation of young patients with Cystic Fibrosis: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>the improved general therapy has led to reduced morbidity and mortality from Cystic Fibrosis (CF), and bone status may have a potentially greater clinical impact.</p> <p>We investigated the correlation between the severity of the clinical condition, bone status and body composition parameters, in a group of children and young adults with CF.</p> <p>Methods</p> <p>we measured lumbar spine bone density and total body composition by dual energy x-ray absorptiometry (DXA) in 82 consecutive CF patients (42 males; median age: 13 years - range: 5-30). Eighty-two healthy subjects, matched for age, gender, height and pubertal stage were recruited as a control group.</p> <p>Results</p> <p>37 patients (45.1%) had a normal bone mineral density (BMD). A BMD reduction were observed in 45 (54.8%) patients. Lumbar spine Z score was positively related to Body Mass Index (BMI) and a higher Shwachman-Kulczycki score, and negatively related to Crispin-Norman score. A positive and significant correlation was also observed between lumbar spine Z score and total body composition.</p> <p>Conclusion</p> <p>a significant BMD reduction can be present early in CF children and adolescents. A careful follow up of bone status is required starting in childhood.</p

Altered Intracellular Localization and Mobility of SBDS Protein upon Mutation in Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients

Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation

We have analysed a family with nine congenital neutropenia patients in four generations, several of which we have studied in a long-term follow-up of over 25 years. The patients were mild to severe neutropenic and suffered from various recurrent bacterial infections. Mutations in the genes ELANE, CSF3R and GFI1 have been reported in patients with autosomal dominant congenital neutropenias. Using a small-scale linkage analysis with markers around the ELANE, CSF3R, CSF3 and GFI1 genes, we were able to determine that the disease segregated with markers around the ELANE gene. We identified a novel mutation in the ELANE gene in all of the affected family members that was not present in any of the healthy family members. The mutation leads to an A28S missense mutation in the mature protein. None of these patients developed leukaemia. This is the first truly multigenerational family with mutations in ELANE as unambiguous cause of severe congenital neutropenia SCN