Respiratory viral infections within one year after pediatric lung transplant.

To characterize epidemiology and risk factors for respiratory viral infections (RVI) in pediatric lung transplant recipients within the first post-transplant year, a retrospective multicenter study of pediatric lung transplant recipients from 1988 to 2005 was conducted at 14 centers in the United States and Europe. Data were recorded for 1 year post transplant. Associations between RVI and continuous and categorical risk factors were assessed using Wilcoxon's rank-sum and chi(2) tests, respectively. Associations between time to RVI and risk factors or survival were assessed by multivariable Cox proportional hazards models. Of 576 subjects, 79 subjects (14%) had 101 RVI in the first year post transplant. Subjects with RVI were younger than those without RVI (median ages 9.7, 13; P<0.01). Viruses detected included adenovirus (n=25), influenza (n=9), respiratory syncytial virus (n=21), parainfluenza virus (n=19), enterovirus (n=4), and rhinovirus (n=22). In a multivariable model for time to first RVI, etiology other than cystic fibrosis (CF), younger age, and no induction therapy were independently associated with risk of RVI. Cytomegalovirus serostatus and acute rejection were not associated with RVI. RVI was independently associated with decreased 12-month survival (hazard ratio 2.6, 95% confidence interval 1.6-4.4). RVI commonly occurs after pediatric lung transplantation with risk factors including younger age and non-CF diagnosis. RVI is associated with decreased 1-year survival.

[Value of sweat conductivity testing in the diagnosis of cystic fibrosis in children].

Objective: To assess the diagnostic value of sweat conductivity testing in Chinese children with cystic fibrosis (CF). Methods: This is a retrospective study. Sweat conductivity tests were conducted in 45 CF children (CF group) and 200 non-CF children (non-CF group) diagnosed with other chronic pulmonary diseases at the No. 2 Department of Respiratory Medicine, Beijing Children's Hospital from May 2014 to June 2018. Pearson's chi-square test was used to assess the differences between CF and non-CF groups. A receiver operating characteristic curve was constructed to calculate the best cut-off value to diagnose or rule out CF. The pulmonary function parameters (forced expiratory volume in the first second, forced vital capacity,forced expiratory flows at 75% of exhaled vital capacity) of CF children over 6 years old were analyzed. The relationship between sweat conductivity and pulmonary function was compared between the two groups (80-120mmol/L vs.>120mmol/L). Results: The age of CF group was 9 (7,12) years old, 19 males (42%) and 26 females(58%); the age of non-CF group was 8 (5,11) years old, 106 males (53%) and 94 females(47%). The results of sweat conductivity test showed that sweat conductivity in CF group 108(99, 122) mmol/L was significantly higher than that in non-CF group 43(36, 52) mmol/L (χ(2)=207, P<0.01). A cut-off value of 80 mmol/L for CF diagnosis showed a sensitivity of 93.3% and a specificity of 98.5%. The receiver operating characteristic curve analysis suggested the best conductivity cut-off value for the diagnosis of CF was at 83.5 mmol/L,with a sensitivity of 93.3% and a specificity of 100%,and an area under the curve of 0.993 (95% confidence interval 0.985-1.000). The best conductivity cut-off value to rule out CF diagnosis was at 63.5 mmol/L,with a sensitivity of 97.8% and a specificity of 90.5%. There was no correlation between the level of sweat conductivity and the extent of pulmonary function decline. Conclusions: Sweat conductivity testing can be used for the screening of CF in Chinese children. A diagnosis of CF should be considered if the value is greater than 80 mmol/L.

Bilateral midshaft femoral fractures in an adolescent baseball player.

Bone disease, specifically low bone mineral density, is a common and undertreated complication that begins during childhood in patients with cystic fibrosis (CF). This case describes a male baseball player, aged 14years, with undiagnosed CF who sustained a left midshaft femoral fracture while running toward base; 8months later, he sustained a right midshaft femoral fracture under similar conditions. After the second fracture, further evaluation revealed low bone mineral density and CF. There is no previously published report of pathologic fractures occurring in the femoral shaft in an athlete with undiagnosed CF. Patients with CF have a higher fracture rate. Low-energy fractures of major bones in athletically active individuals should be viewed with suspicion for an underlying process.

Glycosaminoglycan content in neonatal rat aortic smooth muscle cell cultures.

In the present study the biosynthesis of glycosaminoglycans (GAGs) by neonatal rat aortic smooth muscle cells in culture was studied. Heparan sulfate (HS) was the predominant GAG of the cell layer accounting for 32-49% of the total GAGs depending on the time in culture. The presence of low sulfated chondroitin sulfate (LSC) in aortic smooth muscle cell cultures is reported here for the first time. The effect of ascorbate on the synthesis and accumulation of these macromolecules resulted in a relative increase of C4S and DS in the cell layer. In contrast, the distribution of the GAGs which were secreted into the medium was not significantly effected by the addition of ascorbate. While HS was always found to be a minor component, the other GAGs were present in about equal concentrations. The total GAG accumulation in the medium was much greater (91-97%) than that of the cell layer (3-9%) indicating that the cells are synthesizing relatively large amounts of GAGs, although incorporation of these macromolecules into the extracellular matrix was consistently low.

Congo red binding of elastin in aortic smooth muscle cell cultures.

These studies demonstrate that the strong binding capacity of elastin for Congo red can be used to advantage in aortic smooth muscle cell cultures. A fibrous elastin network fluoresces when Congo red is added. Congo red does not alter accumulation of elastin or of total protein, even when the cells are grown in the presence of the dye for long periods of time, indicating that it is not toxic. Porcine pancreatic elastase was used to solubilize elastin in these cultures, to determine the molar ratio of Congo red to elastin, thus making it possible to estimate the amount of elastin solubilized when the cultures are injured. Congo red binding to elastin will be useful in studying elastin accumulation and/or degradation in vitro and in vivo.

Eosinophilic infiltrates in a pulmonary allograft: a case and review of the literature.

An unusual case of peribronchial eosinophilic infiltrates associated with peripheral blood eosinophilia in a lung transplant patient is described. The role that eosinophils play in lung allograft rejection is reviewed. Tissue eosinophils have been associated with acute pulmonary allograft rejection. Although, eosinophils in bronchoalveolar lavage fluid (BAL) have been observed in allograft rejection, this relationship is less well defined. The role of eosinophils in the pathophysiology of allograft rejection is unclear.

Rationale and design of a randomized trial of home electronic symptom and lung function monitoring to detect cystic fibrosis pulmonary exacerbations: the early intervention in cystic fibrosis exacerbation (eICE) trial.

BACKGROUND: Acute pulmonary exacerbations are central events in the lives of individuals with cystic fibrosis (CF). Pulmonary exacerbations lead to impaired lung function, worse quality of life, and shorter survival. We hypothesized that aggressive early treatment of acute pulmonary exacerbation may improve clinical outcomes. PURPOSE: Describe the rationale of an ongoing trial designed to determine the efficacy of home monitoring of both lung function measurements and symptoms for early detection and subsequent early treatment of acute CF pulmonary exacerbations. STUDY DESIGN: A randomized, non-blinded, multi-center trial in 320 individuals with CF aged 14 years and older. The study compares usual care to a twice a week assessment of home spirometry and CF respiratory symptoms using an electronic device with data transmission to the research personnel to identify and trigger early treatment of CF pulmonary exacerbation. Participants will be enrolled in the study for 12 months. The primary endpoint is change in FEV1 (L) from baseline to 12 months determined by a linear mixed effects model incorporating all quarterly FEV1 measurements. Secondary endpoints include time to first acute protocol-defined pulmonary exacerbation, number of acute pulmonary exacerbations, number of hospitalization days for acute pulmonary exacerbation, time from the end of acute pulmonary exacerbation to onset of subsequent pulmonary exacerbation, change in health related quality of life, change in treatment burden, change in CF respiratory symptoms, and adherence to the study protocol. CONCLUSIONS: This study is a first step in establishing alternative approaches to the care of CF pulmonary exacerbations. We hypothesize that early treatment of pulmonary exacerbations has the potential to slow lung function decline, reduce respiratory symptoms and improve the quality of life for individuals with CF.

Familial neuroendocrine cell hyperplasia of infancy.

BACKGROUND: Neuroendocrine cell hyperplasia of infancy (NEHI) is a recently described children's interstitial lung disease (chILD) disorder of unknown etiology. It manifests clinically with tachypnea, retractions, hypoxemia, and crackles. The characteristic radiographic appearance consists of pulmonary hyperexpansion and ground-glass densities on high-resolution computed tomography (HRCT). Lung histology shows hyperplasia of bombesin-immunopositive neuroendocrine cells within distal bronchioles and alveolar ducts without other identifiable lung pathology or developmental anomaly. METHODS: We describe four families with multiple siblings diagnosed with NEHI. Cases were identified at three pediatric centers. Inclusion criteria included clinical findings consistent with NEHI, lung biopsy confirmation in the index case, and a diagnostic HRCT or biopsy in other siblings. RESULTS: Each family had a proband diagnosed with NEHI based upon pathologic review, and at least one additional sibling diagnosed either by pathologic review or HRCT. All patients presented between 2 and 15 months of age. Both male and female children were affected. The majority of the patients underwent both HRCT and lung biopsy. There were no deaths among affected children. No environmental exposures or other potential etiologies were identified as a cause of presenting symptoms. CONCLUSIONS: The familial occurrence of NEHI suggests the possibility of a genetic etiology for this disorder and highlights the importance of taking a complete family medical history for infants presenting with a suggestive clinical picture. Identification of familial NEHI patients allows for the opportunity to further our understanding of this disorder, its natural history, the phenotypic spectrum, and potential genetic causes.

American Society of Transplantation executive summary on pediatric lung transplantation.

Lung transplantation in children poses distinctly different challenges from those seen in the adult population. This consensus statement reviews the experience in the field of pediatric lung transplantation and highlights areas that deserve further investigation.

CFTR intron 1 increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome.

The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood. The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further. An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc). A second YAC (y5'lucI) was similarly constructed but included a putative positive regulatory element from CFTR intron 1. Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC to assess the role that luc copy number and the presence of intron 1 played in luc expression. The CHO-K1 clonal cell lines demonstrated a wide range of luciferase activity. On average, this activity was significantly higher in clones derived from y5'lucI. After correcting for luc copy number, the presence of intron 1 was still associated with an increase in luciferase activity (P < 0.05), despite the fact that luciferase activity did not correlate with luc copy number in y5'luc-derived clones (r = -0.12). In contrast, the luciferase activity correlated well with luc copy number in the clones derived from y5'luc (r = 0. 75). These data are consistent with a positive role for intron 1 in regulating CFTR expression, but suggest that copy number is not the only factor that determines expression levels, particularly when this element is present. This YAC-based reporter system will provide a unique strategy for further assessment of the cis-acting elements that control CFTR expression.

Skeletal dysplasias and their effect on the respiratory system.

Children with skeletal dysplasia frequently have pulmonary disease which can be life threatening. These pulmonary problems are due to multiple aetiologies including thoracic and craniofacial anomalies predisposing to restrictive lung disease, upper airway obstruction and central apnoea. Recognition of pulmonary disease and early intervention improves the survival and quality of life for these children.

Effect of the reducing environment on the accumulation of elastin and collagen in cultured smooth-muscle cells.

We show here that cultured neonatal-rabbit aortic smooth-muscle cells produce and accumulate significant amounts of insoluble elastin. When grown in the presence of ascorbic acid, the amount of insoluble elastin in these cultures decreases, whereas the accumulation of collagen increases. These changes have been attributed to increased hydroxylation of proline in elastin. The function of ascorbic acid in proline hydroxylation is thought to be that of a reductive cofactor that maintains the proper oxidation state of molecular iron in the enzyme complex. This study shows that both ascorbic and isoascorbic acids act similarly to modify the accumulation of elastin and collagen in culture. On the other hand, cultures grown in the presence of dithiothreitol, a reducing agent previously shown to act as a cofactor for prolyl hydroxylase, do not demonstrate altered elastin accumulation. These studies are consistent with the suggestion that there is a specific role for ascorbic acid in this cellular system that cannot be replaced by other reducing cofactors.

Mammalian cell growth on collagen-hydrogels.

Hydroxyethylmethacrylate (HEMA) hydrogels have a peculiar crater-like topography which renders them ideal for studying cell-to-substrate contact formation. Cultured rabbit aortic smooth muscle cells (SMC) were grown on collagen-HEMA hydrogels, and the ultrastructure of developing cell attachment sites was studied. By 3 hours after cell seeding, both the rounded and spreading SMC appeared anchored to the hydrogel via extra-cellular connective tissue-like material. The fully formed attachment site present at 5-8 day was characterized by large bundles of intracellular myofilaments inserting onto areas of increased electron density along the plasmalemmal membrane. Large amounts of extracellular connective tissue-like material also appeared attached to the areas of increased electron density. Fully formed cell substratum attachment sites were not observed when either elastin-HEMA or hydrogels polymerized in the absence of protein were employed. The growth and collagen synthesis by human embryonic lung fibroblasts (IMR-90) and endothelial cells on collagen-HEMA hydrogels and tissue culture plastic were also examined. Although the two cell types grew equally well on the two surfaces, differences in protein synthesis were observed. The procollagen Type I and III ratio synthesized by the fibroblasts remained the same while the ratio synthesized by the endothelial cells varied even though their morphology appeared similar. The fibroblasts synthesized less collagen when grown on collagen-HEMA hydrogels (this effect may be related to age), while the amount of collagen synthesized by the endothelial cells grown on the two surfaces was similar.

Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture.

The effect of elastase on the extracellular matrix of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Porcine pancreatic elastase was shown to decrease the elastin content in these cultures. Although chemically no distinction could be made between the elastin remaining in the culture matrix after elastase when compared to that in the nontreated cultures, the elastin was dramatically altered morphologically. The elastin assumed a "mottled" appearance after elastase treatment similar to that seen in vivo in emphysema models. A highly sensitive immunogold staining technique was used to detect elastin at the earliest stages of accumulation. Pulse experiments demonstrated an increase in protein synthesis by the cells 20 hr after elastase exposure. The culture system described here provides a model for probing in vivo elastase effects on elastin-containing tissues.

Sleep-disordered breathing in children with achondroplasia.

OBJECTIVE: Our objective was to characterize sleep-disordered breathing in 88 children with achondroplasia aged 1 month to 12.6 years. RESULTS: At the time of their initial polysomnography, five children had previously undergone tracheostomy, and seven children required supplemental oxygen. Initial polysomnography demonstrated a median obstructive apnea index of 0 (range, 0 to 19.2 apneas/hr). The median number of central apneas with desaturation per study was 0.5 (0 to 49), the median oxygen saturation nadir was 91% (50% to 99%), and the median peak end-tidal pCO2 was 47 mm Hg (36 to 87 mm Hg). Forty-two children (47.7%) had abnormal initial study results, usually caused by hypoxemia. Two children with severe obstructive sleep apnea eventually required continuous positive airway pressure therapy, and three additional children required tracheostomies. CONCLUSIONS: (1) Children with achondroplasia often have sleep-related respiratory disturbances, primarily hypoxemia. (2) The majority do not have significant obstructive or central apnea; however, a substantial minority are severely affected. (3) Tonsillectomy and adenoidectomy decreases the degree of upper airway obstruction in most but not all children with achondroplasia and obstructive sleep apnea. (4) Restrictive lung disease can present at a young age in children with achondroplasia.

An unusual case of fatal pulmonary allograft rejection.

This case report describes an atypical form of acute pulmonary allograft rejection that was refractory to conventional therapy. The rejection manifested primarily as interstitial lymphocytic infiltrates with little perivascular involvement. Despite aggressive therapy the patient died within 7 months of transplant. The timely recognition and treatment of unusual forms of allograft rejection is vital in the management of pulmonary transplant patients.

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells.

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells.

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.