Development Of Chronic Lung Disease Research Articles

Nrf2 deficiency exacerbated pulmonary pyroptosis in maternal hypoxia-induced intrauterine growth restriction offspring mice

Maternal prenatal hypoxia is an important contributor to intrauterine growth restriction (IUGR), which impedes fetal lung maturation and leads to the development of chronic lung diseases. Although evidence suggests the involvement of pyroptosis in IUGR, the molecular mechanism of pyroptosis is still unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been found to potentially interact with gasdermin D (GSDMD), the key protein responsible for pyroptosis, indicating its crucial role in inhibiting pyroptosis. Therefore, we hypothesized that Nrf2 deficiency is a key molecular responsible for lung pyroptosis in maternal hypoxia-induced IUGR offspring mice. Pregnant WT and Nrf2-/- mice were exposed to hypoxia (10.5% O2) to mimic IUGR model. We assessed body weight, lung histopathology, pulmonary angiogenesis, oxidative stress levels, as well as mRNA and protein expressions related to inflammation in the 2-week-old offspring. Additionally, we conducted a dual-luciferase reporter assay to confirm the targeting relationship between Nrf2 and GSDMD. Our findings revealed that offspring with maternal hypoxia-induced IUGR exhibited reduced birth weight, catch-up growth delay, and pulmonary dysplasia. Furthermore, we observed impaired nuclear translocation of Nrf2 and increased GSDMD-mediated pyroptosis in these offspring with IUGR. Moreover, the dual-luciferase reporter assay demonstrated that Nrf2 could directly inhibit GSDMD transcription; deficiency of Nrf2 exacerbated pyroptosis and pulmonary dysplasia in offspring with maternal hypoxia-induced IUGR. Collectively, our findings suggest that Nrf2 deficiency induces GSDMD-mediated pyroptosis and pulmonary dysplasia in offspring with maternal hypoxia-induced IUGR; thus highlighting the potential therapeutic approach of targeting Nrf2 for treating prenatal hypoxia-induced pulmonary dysplasia in offspring.

Pleural Effusion and Chylothorax in Congenital Diaphragmatic Hernia-Risk Factors, Management and Outcome.

Background: Pleural effusion and chylothorax are common complications in the treatment of congenital diaphragmatic hernia (CDH). We set out to identify risk factors for chylothorax development in patients with CDH and to investigate the association of pleural effusion and chylothorax with neonatal morbidity and mortality. Methods: In this retrospective cohort study, we included 396 neonates with CDH treated at our institution between January 2013 and June 2019. Preoperative and postoperative chest radiographs and clinical data were evaluated and correlated with morbidity, complications and mortality. Results: Laboratory-confirmed chylothorax occurred in 58 (18.6%) of all CDH cases. Pleural effusion was frequently observed as a postoperative complication but also occurred as a pre-existing condition. Neonates with large defects of size C and D, patch repair, the need for presurgical and/or postsurgical ECMO support, pulmonary hypertension, liver-up phenomenon and lower relative fetal lung volume were associated with higher occurrences of chylothorax. After stepwise logistic regression, larger CDH defects (p < 0.0001) and the need for postsurgical ECMO (p = 0.0158) remained significant risk factors for CTX to occur (AUC 0.71). The same potential risk factors were used to assess their association with both presurgical and postsurgical pleural effusion. After stepwise logistic regression, only the need for presurgical ECMO remained significantly associated with presurgical PE (p < 0.01, AUC 0.65) and patch repair as the therapeutic intervention remained significantly associated with the occurrence of postsurgical PE (p < 0.0001, AUC 0.80). Patients with CTX had longer durations of both MV (p < 0.0001) and subsequent ventilatory assistance with spontaneous breathing (p = 0.0004), increased total lengths of hospitalization (p < 0.0001), increased durations of ECMO (p < 0.01) and increased incidences of CLD (p < 0.0001) compared to patients without CTX. No significant difference could be found for survival in both groups (p = 0.12). Conclusions: Our data suggest that the incidence of chylothorax is associated with large diaphragmatic defects, the need for postsurgical ECMO and the development of chronic lung disease, but not with survival.

Fetal MRI-Based Mediastinal Shift Angle (MSA) and Percentage Area of Left Ventricle (pALV) as Prognostic Parameters for Congenital Diaphragmatic Hernia.

Fetal magnetic resonance imaging (MRI) is broadly used as a method for assessing prognosis in congenital diaphragmatic hernia (CDH). In addition to the extent of lung hypoplasia, determined by measuring the lung volume, cardiac impairment due to pulmonary hypertension and left cardiac hypoplasia is decisive for the prognosis. The percentage area of left ventricle (pALV) describes the percentage of the inner area of the left ventricle in relation to the total area, whereas the mediastinal shift angle (MSA) quantifies the extent of cardiac displacement. The prognostic value of pALV and MSA should be evaluated in terms of survival, the need for extracorporeal membrane oxygenation (ECMO) therapy, and the development of chronic lung disease (CLD). In a total of 122 fetal MRIs, the MSA and pALV were measured retrospectively and complete outcome parameters were determined regarding survival for all 122 subjects, regarding ECMO therapy in 109 cases and about the development of CLD in 78 cases. The prognostic value regarding the endpoints was evaluated using logistic regression and ROC analysis. The MSA was significantly higher in children who received ECMO therapy (p = 0.0054), as well as in children who developed CLD (p = 0.0018). ROC analysis showed an AUC of 0.68 for ECMO requirement and 0.77 with respect to CLD development. The pALV showed a tendency towards higher levels in children who received ECMO therapy (p = 0.0824). The MSA and the pALV had no significant effect on survival (MSA: p = 0.4293, AUC = 0.56; pALV: p = 0.1134, AUC = 0.57). The MSA determined in fetal MRI is a suitable prognostic parameter for ECMO requirement and CLD development in CDH patients and can possibly be used as a supplement to the established parameters.

Neuroimmune Interactions in the Lung.

Barrier tissues are highly innervated by sensory and autonomic nerves that are positioned in close proximity to both stromal and immune cell populations. Together with a growing awareness of the far-reaching consequences of neuroimmune interactions, recent studies have uncovered key mechanisms through which they contribute to organ homeostasis and immunity. It has also become clear that dysregulation of such interactions is implicated in the development of chronic lung diseases. This review describes the characteristics of the lung nervous system and discusses the molecular mechanisms that underlie lung neuroimmune interactions in infection and disease. We have contextualized the current literature and identified opportune areas for further investigation. Indeed, both the lung-brain axis and local neuroimmune interactions hold enormous potential for the exploration and development of novel therapeutic strategies targeting lung diseases. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Nickel-induced transcriptional memory in lung epithelial cells promotes interferon signaling upon nicotine exposure

Exposure to nickel, an environmental respiratory toxicant, is associated with lung diseases including asthma, pulmonary fibrosis, bronchitis and cancers. Our previous studies have shown that a majority of the nickel-induced transcriptional changes are persistent and do not reverse even after the termination of exposure. This suggested transcriptional memory, wherein the cell ‘remembers’ past nickel exposure. Transcriptional memory, due to which the cells respond more robustly to a previously encountered stimulus has been identified in a number of organisms. Therefore, transcriptional memory has been described as an adaptive mechanism. However, transcriptional memory caused by environmental toxicant exposures has not been well investigated. Moreover, how the transcriptional memory caused by an environmental toxicant might influence the outcome of exposure to a second toxicant has not been explored. In this study, we investigated whether nickel-induced transcriptional memory influences the outcome of the cell’s response to a second respiratory toxicant, nicotine. Nicotine, an addictive compound in tobacco, is associated with the development of chronic lung diseases including chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Our results show that nicotine exposure upregulated a subset of genes only in the cells previously exposed to nickel. Furthermore, our analyses indicate robust activation of interferon (IFN) signaling in these cells. IFN signaling is a driver of inflammation, which is associated with many chronic lung diseases. Therefore, our results suggest that nicotine exposure of lung cells that retain the transcriptional memory of previous nickel exposure could result in increased susceptibility to developing chronic inflammatory lung diseases.

Understanding the Under-Recognised Burden and Unmet Needs in Infants and Children with Respiratory Syncytial Virus

For this article, an interview was conducted by EMJ in June 2023 with key opinion leader, Lindsay Broadbent, Lecturer (Assistant Professor) in Virology in the Faculty of Health and Medical Sciences at the University of Surrey, Guildford, UK. Broadbent specialises in respiratory virus–host interactions and subsequent innate immune responses, and the role of respiratory viruses in longer-term lung damage and the development of chronic lung disease. In this interview, Broadbent discussed the global burden of respiratory syncytial virus (RSV), a virus spread by micro-droplets. Broadbent highlighted RSV as a major cause of hospitalisations, particularly in children under the age of 1 year, which can impose a significant burden on health, particularly in infants, and healthcare systems globally. Broadbent provided insights into the leading risk factors of RSV, and emphasised the need for preventative measures to mitigate severe infections. Additionally, Broadbent highlighted the importance of improving the diagnosis and treatment options to address the unmet medical needs related with RSV. They called for increased awareness of RSV, and understanding of why certain infants are more susceptible to severe disease.

Prenatal vitamin D supplementation mitigates inflammation-related alveolar remodeling in neonatal mice.

The development of chronic lung disease in the neonate, also known as bronchopulmonary dysplasia (BPD), is the most common long-term complication in prematurely born infants. In BPD, the disease-characteristic inflammatory response culminates in nonreversible remodeling of the developing gas exchange area, provoked by the impact of postnatal treatments such as mechanical ventilation (MV) and oxygen treatment. To evaluate the potential of prenatal treatment regimens to modulate this inflammatory response and thereby impact the vulnerability of the lung toward postnatal injury, we designed a multilayered preclinical mouse model. After administration of either prenatal vitamin D-enriched (VitD+; 1,500 IU/g food) or -deprived (VitD-; <10 IU/kg) food during gestation in C57B6 mice (the onset of mating until birth), neonatal mice were exposed to hyperoxia (FiO2 = 0.4) with or without MV for 8 h at days 5-7 of life, whereas controls spontaneously breathed room air. Prenatal vitamin D supplementation resulted in a decreased number of monocytes/macrophages in the neonatal lung undergoing postnatal injury together with reduced TGF-β pathway activation. In consequence, neonatal mice that received a VitD+ diet during gestation demonstrated less extracellular matrix (ECM) remodeling upon lung injury, reflected by the reduction of pulmonary α-smooth muscle actin-positive fibroblasts, decreased collagen and elastin deposition, and lower amounts of interstitial tissue in the lung periphery. In conclusion, our findings support strategies that attempt to prevent vitamin D insufficiency during pregnancy as they could impact lung health in the offspring by mitigating inflammatory changes in neonatal lung injury and ameliorating subsequent remodeling of the developing gas exchange area.NEW & NOTEWORTHY Vitamin D-enriched diet during gestation resulted in reduced lung inflammation and matrix remodeling in neonatal mice exposed to clinically relevant, postnatal injury. The results underscore the need to monitor the subclinical effects of vitamin D insufficiency that impact health in the offspring when other risk factors come into play.

SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SCV2), which has resulted in higher morbidity and mortality rate than other respiratory viral infections, such as Influenza A virus (IAV) infection. Investigating the molecular mechanisms of SCV2-host infection vs IAV is vital in exploring antiviral drug targets against SCV2. We assessed differential gene expression in human nasal cells upon SCV2 or IAV infection using RNA sequencing. Compared to IAV, we observed alterations in both metabolic and cytoskeletal pathways suggestive of epithelial remodeling in the SCV2-infected cells, reminiscent of pathways activated as a response to chronic injury. We found that spike protein interaction with the epithelium was sufficient to instigate these epithelial responses using a SCV2 spike pseudovirus. Specifically, we found downregulation of the mitochondrial markers SIRT3 and TOMM22. Moreover, SCV2 spike infection increased extracellular acidification and decreased oxygen consumption rate in the epithelium. In addition, we observed cytoskeletal rearrangements with a reduction in the actin-severing protein cofilin-1 and an increase in polymerized actin, indicating epithelial cytoskeletal rearrangements. This study revealed distinct epithelial responses to SCV2 infection, with early mitochondrial dysfunction in the host cells and evidence of cytoskeletal remodeling that could contribute to the worsened outcome in COVID-19 patients compared to IAV patients. These changes in cell structure and energetics could contribute to cellular resilience early during infection, allowing for prolonged cell survival and potentially paving the way for more chronic symptoms. IMPORTANCE COVID-19 has caused a global pandemic affecting millions of people worldwide, resulting in a higher mortality rate and concerns of more persistent symptoms compared to influenza A. To study this, we compare lung epithelial responses to both viruses. Interestingly, we found that in response to SARS-CoV-2 infection, the cellular energetics changed and there were cell structural rearrangements. These changes in cell structure could lead to prolonged epithelial cell survival, even in the face of not working well, potentially contributing to the development of chronic symptoms. In summary, these findings represent strategies utilized by the cell to survive the infection but result in a fundamental shift in the epithelial phenotype, with potential long-term consequences, which could set the stage for the development of chronic lung disease or long COVID-19.

The association between sarcopenia and incident chronic lung disease in the general population: A longitudinal study based on CHARLS data

BackgroundData regarding the association of sarcopenia with chronic lung disease (CLD) has led to inconclusive results. The main goal of this research was to investigate the association between sarcopenia and CLD in middle-aged and elderly individuals in China. MethodsThe study sample consisted of 11,077 individuals without CLD at baseline chosen from the China Health and Retirement Longitudinal Study (CHARLS) data from 2015, followed up until 2018. Sarcopenia was identified utilizing the criteria set by the Asian Working Group on Sarcopenia (AWGS 2019) in 2019. Individuals were categorized into no-sarcopenia, possible-sarcopenia, and sarcopenia groups. The outcome of the study was considered to be incident CLD, which included chronic bronchitis, emphysema, pulmonary heart disease, and asthma. The association between sarcopenia and the risk of CLD was also examined by employing weighted Cox proportional hazard regression models. ResultsA total of 356 (3.20 %) participants developed CLD during the 3.6-year follow-up period. The cumulative incidence of CLD in the no-sarcopenia, possible-sarcopenia, and sarcopenia groups was 2.80 % (230/8222), 4.37 % (55/1260), and 4.45 % (71/1595), respectively. Individuals with possible sarcopenia {hazard ratio [HR] [95 % confidence interval (CI)]: 1.48 [1.04–2.09]} and sarcopenia [HR (95 % CI): 1.68 (1.12–2.51)] demonstrated a considerably high risk of developing CLD compared to individuals in the no-sarcopenia group. Moreover, individuals diagnosed with sarcopenia, as per the criteria established by the European Working Group on Sarcopenia in Older People (EWGSOP) 2018, were at considerably high risk for developing CLD compared to those in the no-sarcopenia group. ConclusionThis research involving adult Chinese individuals demonstrated a significant association between, possible sarcopenia and sarcopenia with an elevated risk of incident CLD, thereby emphasizing the importance of monitoring respiratory health in this population. Key pointsQuestion: Whether muscle mass and sarcopenia are associated with the development of chronic lung disease (CLD) in Asian middle-aged and elderly individuals.Findings: This longitudinal study encompassing 11,077 adults aged ≥45 years from the China Health and Retirement Longitudinal Study (CHARLS) data with 3.6 years of follow-up revealed a positive association between sarcopenia at baseline and incidence of CLD.Meaning: The findings suggest that possible sarcopenia and sarcopenia are linked to the development of CLD. Consequently, middle-aged and elderly individuals with possible sarcopenia and sarcopenia can be considered vulnerable regarding the primary prevention strategies for CLD.

Microcomputed tomography visualization and quantitation of the pulmonary arterial microvascular tree in mouse models of chronic lung disease.

Pulmonary vascular dysfunctionis characterized by remodeling and loss of microvessels in the lung and is a major manifestation of chronic lung diseases (CLD). In murine models of CLD, the small arterioles and capillaries are the first and most prevalent vessels that are affected by pruning and remodeling. Thus, visualization of the pulmonary arterial vasculature in three dimensions is essential to define pruning and remodeling both temporally and spatially and its role in the pathogenesis of CLD, aging, and tissue repair. To this end, we have developed a novel method to visualize and quantitate the murine pulmonary arterial circulation using microcomputed tomography(µCT) imaging. Using this perfusion technique, we can quantitate microvessels to approximately 6 µM in diameter. We hypothesize that bleomycin-induced injury would have a significant impact on the arterial vascular structure. As proof of principle, we demonstrated that as a result of bleomycin-induced injury at peak fibrosis, significant alterations in arterial vessel structure were visible in the three-dimensional models as well as quantification. Thus, we have successfully developed a perfusion methodology and complementary analysis techniques, which allows for the reconstruction, visualization, and quantitation of the mouse pulmonary arterial microvasculature in three-dimensions. This tool will further support the examination and understanding of angiogenesis during the development of CLD as well as repair following injury.

"Usefulness of the Acute Phase Reactants (APR) score (Part 2): Characteristics of the APR-Score in Wilson-Mikity Syndrome"

The etiology of Wilson-Mikity syndrome (WMS) remains uncertain. Inflammation of the fetal lung caused by intrauterine infection spilling over into the lungs results in lung tissue collapse and birth before complete repair is achieved. Continued spontaneous breathing exacerbates lung damage that results in the development of chronic lung disease.

Commensal bacteria of the lung microbiota synergistically inhibit inflammation in a three-dimensional epithelial cell model.

Patients with chronic lung disease suffer from persistent inflammation and are typically colonized by pro-inflammatory pathogenic bacteria. Besides these pathogens, a wide variety of commensal species is present in the lower airways but their role in inflammation is unclear. Here, we show that the lung microbiota contains several species able to inhibit activation of the pro-inflammatory NF-κB pathway and production of interleukin 8 (IL-8), triggered by lipopolysaccharide (LPS) or H2O2, in a physiologically relevant three-dimensional (3D) lung epithelial cell model. We demonstrate that the minimal dose needed for anti-inflammatory activity differs between species (with the lowest dose needed for Rothia mucilaginosa), and depends on the type of pro-inflammatory stimulus and read out. Furthermore, we evaluated synergistic activity between pairs of anti-inflammatory bacteria on the inhibition of the NF-κB pathway and IL-8 secretion. Synergistic anti-inflammatory activity was observed for 4/10 tested consortia. These findings indicate that various microbiota members can influence lung inflammation either alone or as a consortium. This information can contribute to a better understanding of the lung microbiota in chronic lung disease development and process, and could open up new avenues for treatment.

Chronic lung disease in Saudi preterm infants

Background: Despite advances in neonatal medicine, chronic lung disease (CLD) remains a major cause of pulmonary morbidity in preterm infants. Objective: To determine the prevalence of CLD and to identify the possible risk factors that may predict the development of CLD. Patients and Methods: A retrospective study of preterm infants admitted to the NICU between July 2011 and June 2022 with gestational age (GA) < 32 weeks and birth weight (BW) < 1500 g was performed. Results: A total of 706 preterm infants were included in the study, of whom 202 (28.6%) infants developed CLD. Of the 202 preterm infants who developed CLD, 194 survivors; 93 (47.9%) had mild, 58 (29.9%) had moderate and 43 (22.2%) had severe CLD. Multivariate analysis showed that low GA (odds ratio [OR]: 0.62; 95% confidence interval [CI]: 0.43-0.98), low BW (OR: 20.6; 95% CI: 17.1-29.3), use of mechanical ventilation (OR: 1.07; 95% CI: 0.87-1.54), higher peak inspiratory pressure (PIP) (OR: 1.48; 95% CI: 1.44-1.91), higher fraction of inspired oxygen (FiO2) use (OR: 0.11; 95% CI: 0.05-0.19), duration of mechanical ventilation (OR: 3.56; 95% CI: 4.28-3.75) and frequent blood transfusion (OR: 0.65; 95% CI: 0.53-0.87) were identified as the main risk factors for the development of CLD. Conclusions: The prevalence of CLD among preterm infants was 28.6%. The most relevant predictors of CLD were GA, BW, mechanical ventilation, higher PIP, higher FiO2 use, duration of mechanical ventilation and frequent blood transfusion.

Insufficient Syndecan-4 is associated with chronic lung disease development in preterm infants.

Insufficient Syndecan-4 is associated with chronic lung disease development in preterm infants.

Automated Particle Analysis Using Field-Emission Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) to Characterize Inhaled Particulate Matter (PM) in Biopsied Lung Tissue

Abstract The role of inhaled particulate matter in the development of chronic lung disease in military veterans remains ill-defined and controversial. Characterizing the nature of such inhaled particulates in surgical lung biopsy tissue is an important step in evaluating the association between exposure and illness. We have developed a protocol for automated in-situ analysis of inorganic inhaled particulate matter using field-emission scanning electron microscopy with energy dispersive X-ray spectroscopy. Over 120,000 particles of both a geological and manufactured nature were analyzed and characterized according to their chemistry and morphology. The most abundant particles were clays, feldspars, silica, titanium dioxides, and metals and were by and large less than 2.5 µm in the greatest dimension. Automating the process allows for efficient in-situ analysis of thousands of particles each day and the development of large datasets.

Loss of E-cadherin is causal to pathologic changes in chronic lung disease

Epithelial cells line the lung mucosal surface and are the first line of defense against toxic exposures to environmental insults, and their integrity is critical to lung health. An early finding in the lung epithelium of patients with chronic obstructive pulmonary disease (COPD) is the loss of a key component of the adherens junction protein called E-cadherin. The cause of this decrease is not known and could be due to luminal insults or structural changes in the small airways. Irrespective, it is unknown whether the loss of E-cadherin is a marker or a driver of disease. Here we report that loss of E-cadherin is causal to the development of chronic lung disease. Using cell-type-specific promoters, we find that knockout of E-cadherin in alveolar epithelial type II but not type 1 cells in adult mouse models results in airspace enlargement. Furthermore, the knockout of E-cadherin in airway ciliated cells, but not club cells, increase airway hyperreactivity. We demonstrate that strategies to upregulate E-cadherin rescue monolayer integrity and serve as a potential therapeutic target.

Blood Adipokines/Cytokines in Young People with Chronic Bronchitis and Abdominal Obesity.

The pathogenesis of the development of chronic lung diseases assumes the participation of systemic inflammation factors, as well as hormone-like substances produced by adipose tissue. The aim of this study was to evaluate the associations of certain adipokines/cytokines and chronic bronchitis against the background of abdominal obesity in young people. The study included 1415 people aged 25−44. In total, 115 people were selected by the random numbers method, who were divided into two subgroups: those with chronic bronchitis and abdominal obesity and those with chronic bronchitis without abdominal obesity. A control group of patients with comparable gender and age was also selected. In the group of patients with chronic bronchitis, adiponectin, TNFa and GIP levels were 1.4 times higher. The levels of C-peptide, MCP-1 and PP in the group of chronic bronchitis were 1.3 times higher compared to the control. Adipsin, lipocalin-2, IL-6 and resistin were significantly higher in the group with chronic bronchitis. Glucagon, amylin and ghrelin were 2.2, 2.3 and 3.2 times lower, respectively, in the group of patients with chronic bronchitis. Against the background of abdominal obesity, the probability of having chronic bronchitis increased with an increase in the level of lipocalin-2 and GIP and TNFa.

Follow-up study of infants recruited to the randomised, placebo-controlled trial of azithromycin for the prevention of chronic lung disease of prematurity in preterm infants—study protocol for the AZTEC-FU study

BackgroundPreterm birth, especially at less than 30 weeks’ gestation, is significantly associated with respiratory, neurodevelopmental and growth abnormalities. The AZTEC study has recruited 799 infants born at < 30 weeks’ gestation to determine if a ten-day intravenous treatment with azithromycin improves survival without development of chronic lung disease of prematurity (CLD) at 36 weeks’ post menstrual age (PMA) when compared to placebo. The follow-up studies will compare respiratory, neurodevelopmental and growth outcomes up to 2 years of corrected age between infants who received azithromycin and those who received placebo in the early neonatal period.MethodsSurvivors at 36 weeks’ PMA from the main Azithromycin Therapy for Chronic Lung Disease of Prematurity (AZTEC) study with parental consent will continue to be followed up to discharge from the neonatal unit and to 2 years of corrected age. Length of stay, rates of home oxygen, length of supplemental oxygen requirement, hospital admissions, drug usage, respiratory illness, neurodevelopmental disability and death rates will be reported. Data is being collected via parentally completed respiratory and neurodevelopmental questionnaires at 1 and 2 years of corrected age respectively. Additional information is being obtained from various sources including hospital discharge and clinical letters from general practitioners and hospitals as well as from national databases including the National Neonatal Research Database and NHS Digital.DiscussionThe AZTEC-FU study will assess mortality and important neonatal morbidities including respiratory, neurodevelopmental and growth outcomes. Important safety data will also be collected, including the incidence of potential consequences of early macrolide use, primarily pyloric stenosis. This study may have implications on future neonatal care.Trial registrationThe study was retrospectively registered on ISRCTN (ISRCTN47442783).

AZTEC—azithromycin therapy for prevention of chronic lung disease of prematurity: a statistical analysis plan for clinical outcomes

BackgroundThe AZTEC trial is a multi-centre, randomised, placebo-controlled trial of azithromycin to improve survival without development of chronic lung disease of prematurity (CLD) in preterm infants. The statistical analysis plan for the clinical outcomes of the AZTEC trial is described.Methods and designA double-blind, randomised, placebo-controlled trial of a 10-day course of intravenous azithromycin (20 mg/kg for 3 days; 10 mg/kg for 7 days) administered to preterm infants born at < 30 weeks’ gestational age across UK tertiary neonatal units. Following parental consent, infants are randomly allocated to azithromycin or placebo, with allocated treatment starting within 72 h of birth. The primary outcome is survival without moderate/severe CLD at 36 weeks’ postmenstrual age (PMA). Serial respiratory fluid and stool samples are being collected up to 21 days of life. The target sample size is 796 infants, which is based on detecting a 12% absolute difference in survival without moderate/severe CLD at 36 weeks’ PMA (90% power, two-sided alpha of 0.05) and includes 10% loss to follow-up.ResultsBaseline demographic and clinical characteristics will be summarised by treatment arm and in total. Categorical data will be summarised by numbers and percentages. Continuous data will be summarised by mean, standard deviation, if data are normal, or median, interquartile range, if data are skewed. Tests of statistical significance will not be undertaken for baseline characteristics. The primary analysis, on the intention to treat (ITT) population, will be analysed using multilevel logistic regression, within a multiple imputation framework. Adjusted odds ratios, 95% confidence intervals, and p-values will be presented. For all other analyses, the analysis population will be based on the complete case population, which is a modified ITT population. All analyses will be adjusted for gestational age and treatment arm and account for any clustering by centre and/or multiple births as a random effect.ConclusionWe describe the statistical analysis plan for the AZTEC trial, including the analysis principles, definitions of the key clinical outcomes, methods for primary analysis, pre-specified subgroup analysis, sensitivity analysis, and secondary analysis. The plan has been finalised prior to the completion of recruitment.Trial registrationISRCTN registry ISRCTN11650227. Registered on 31 July 2018.

Cellular mechanisms involved in the pathogenesis of airway remodeling in chronic lung disease

ABSTRACT Background Airway epithelial cells and lung fibroblasts play an important role in the development of chronic lung disease, but the exact mechanisms responsible have not been clarified. Our objective was to investigate the involvement of these cells in the inflammatory response associated to chronic lung disease. Methods Human lung fibroblasts and airway epithelial cells were challenged with Interleukin-1β and hypoxia, and with inhibitory (simvastatin) stimuli of the inflammatory response. Expression of markers of local inflammation ((IL-8, monocyte chemoattractant protein-1 (MCP-1), factor-κB1 (NF-κB1)), systemic inflammation ((C-reactive protein (CRP) and serum amyloid A (SAA)) and proteases matrix metalloproteinase (MMP) 9 and 12 were assessed by PCR and ELISA. Apoptosis/necrosis was analyzed by flow cytometry. Results Our results showed that the lung fibroblasts had a higher expression of local and systemic inflammation and protease activity markers when they were treated with IL-1β compared to airway epithelial cells. Under hypoxic conditions, we observed a decrease in systemic inflammation in lung fibroblasts, which was further attenuated by simvastatin. Conclusion The lung fibroblasts seem to be the main initially stimulated cells that could potentially trigger the inflammatory response, and be responsible for the eventual onset of chronic lung disease. The involvement of IL-1ß stimulation in systemic inflammatory and proteinase imbalance biomarkers is higher in lung fibroblasts. Apoptosis is not a predominant mechanism in these cells.