Search

Martin Garcia-Ryde,1 Nicole MD van der Burg,1 Carin E Larsson,1 Anna-Karin Larsson-Callerfelt,2 Gunilla Westergren-Thorsson,2 Leif Bjermer,1 Ellen Tufvesson1 1Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, Lund, Sweden; 2Department of Experimental Medical Science, Lung Biology, Lund University, Lund, SwedenCorrespondence: Ellen Tufvesson, Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, BMC, D12, 221 84, Lund, Sweden, Email Ellen.Tufvesson@med.lu.seBackground and aim: Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (COPD) but more mechanistic studies are needed. Cigarette smoke extract (CSE) can elicit a strong response in many COPD-related cell types, but no studies have been performed in lung fibroblasts. Therefore, we aimed to investigate the effect of CSE on gene expression in lung fibroblasts from healthy and COPD subjects.Patients and methods: Primary lung fibroblasts, derived from six healthy and six COPD subjects (all current or ex-smokers), were either unstimulated (baseline) or stimulated with 30% CSE for 4 h prior to RNA isolation. The mRNA expression levels were measured using the NanoString nCounter Human Fibrosis V2 panel (760 genes). Pathway enrichment was assessed for unique gene ontology terms of healthy and COPD.Results: At baseline, a difference in the expression of 17 genes was found in healthy and COPD subjects. Differential expression of genes after CSE stimulation resulted in significantly less changes in COPD lung fibroblasts (70 genes) than in healthy (207 genes), with 51 genes changed in both. COPD maintained low NOTCH signaling throughout and upregulated JUN > 80%, indicating an increase in apoptosis. Healthy downregulated the Mitogen-activated protein kinase (MAPK) signaling cascade, including a ≥ 50% reduction in FGF2, CRK, TGFBR1 and MEF2A. Healthy also downregulated KAT6A and genes related to cell proliferation, all together indicating possible cell senescence signaling.Conclusion: Overall, COPD lung fibroblasts responded to CSE stimulation with a very different and deficient expression profile compared to healthy. Highlighting that stimulated healthy cells are not an appropriate substitute for COPD cells which is important when investigating the mechanisms of COPD.Plain Language Summary: We investigated, for the first time, the effect of cigarette smoke extract (CSE) on gene expression in lung fibroblasts from healthy subjects and subjects with COPD. We found that fibroblasts from subjects with COPD respond very differently than from healthy subjects by changing fewer gene expressions after CSE. Pathway enrichment suggested that fibroblasts from subjects with COPD respond to CSE by affecting genes involved in apoptosis pathways, while fibroblasts from healthy subjects respond by affecting genes involved in cell senescence. This study highlights the importance of assessing stimulated COPD cells in mechanistic studies.Keywords: COPD, lung fibroblast, gene expression, cigarette smoke extract, GO term, signaling pathways

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

Introduction: Chronic lung disorders involve pathological alterations in the lung tissue with hypoxia as a consequence. Hypoxia may influence the release of inflammatory mediators and growth factors including vascular endothelial growth factor (VEGF) and prostaglandin (PG)E 2 . The aim of this work was to investigate how hypoxia affects human lung epithelial cells in combination with profibrotic stimuli and its correlation to pathogenesis. Methods: Human bronchial (BEAS-2B) and alveolar (hAELVi) epithelial cells were exposed to either hypoxia (1% O 2 ) or normoxia (21% O 2 ) during 24 h, with or without transforming growth factor (TGF)-β1. mRNA expression of genes and proteins related to disease pathology were analysed with qPCR, ELISA or immunocytochemistry. Alterations in cell viability and metabolic activity were determined. Results: In BEAS-2B and hAELVi, hypoxia significantly dowregulated genes related to fibrosis, mitochondrial stress, oxidative stress, apoptosis and inflammation whereas VEGF receptor 2 increased. Hypoxia increased the expression of Tenascin-C, whereas both hypoxia and TGF-β1 stimuli increased the release of VEGF, IL-6, IL-8 and MCP-1 in BEAS-2B. In hAELVi, hypoxia reduced the release of fibroblast growth factor, epidermal growth factor, PGE 2 , IL-6 and IL-8, whereas TGF-β1 stimulus significantly increased the release of PGE 2 and IL-6. TGF-β1 stimulated BEAS-2B cells showed a decreased release of VEGF-A and IL-8, while TGF-β1 stimulated hAELVi cells showed a decreased release of PGE 2 and IL-8 during hypoxia compared to normoxia. Metabolic activity was significantly increased by hypoxia in both epithelial cell types. Discussion: In conclusion, our data indicate that bronchial and alveolar epithelial cells respond differently to hypoxia and profibrotic stimuli. The bronchial epithelium appears more responsive to changes in oxygen levels and remodelling processes compared to the alveoli, suggesting that hypoxia may be a driver of pathogenesis in chronic lung disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Berggren-Nylund, Ryde, Löfdahl, Ibáñez-Fonseca, Kåredal, Westergren-Thorsson, Tufvesson and Larsson-Callerfelt.)

Gene therapy approaches using viral vectors for the overexpression of target genes have been for several years the focus of gene therapy research against neurological disorders. These approaches deliver robust expression of therapeutic genes, but are typically limited to the delivery of single genes and often do not manipulate the expression of the endogenous locus. In the last years, the advent of CRISPR-Cas9 technologies have revolutionized many areas of scientific research by providing novel tools that allow simple and efficient manipulation of endogenous genes. One of the applications of CRISPR-Cas9, termed CRISPRa, based on the use of a nuclease-null Cas9 protein (dCas9) fused to transcriptional activators, enables quick and efficient increase in target endogenous gene expression. CRISPRa approaches are varied, and different alternatives exist with regards to the type of Cas9 protein and transcriptional activator used. Several of these approaches have been successfully used in neurons in vitro and in vivo , but have not been so far extensively applied for the overexpression of genes involved in synaptic transmission. Here we describe the development and application of two different CRISPRa systems, based on single or dual Lentiviral and Adeno-Associated viral vectors and VP64 or VPR transcriptional activators, and demonstrate their efficiency in increasing mRNA and protein expression of the Cnr1 gene, coding for neuronal CB1 receptors. Both approaches were similarly efficient in primary neuronal cultures, and achieved a 2-5-fold increase in Cnr1 expression, but the AAV-based approach was more efficient in vivo. Our dual AAV-based VPR system in particular, based on Staphylococcus aureus dCas9, when injected in the hippocampus, displayed almost complete simultaneous expression of both vectors, high levels of dCas9 expression, and good efficiency in increasing Cnr1 mRNA as measured by in situ hybridization. In addition, we also show significant upregulation of CB1 receptor protein in vivo , which is reflected by an increased ability in reducing neurotransmitter release, as measured by electrophysiology. Our results show that CRISPRa techniques could be successfully used in neurons to target overexpression of genes involved in synaptic transmission, and can potentially represent a next-generation gene therapy approach against neurological disorders., (Copyright © 2020 Di Maria, Moindrot, Ryde, Bono, Quintino and Ledri.)

Objectives: The objectives of this study were to measure 'health-related quality of life' (HRQoL) in children following slide tracheoplasty for long-segment tracheal stenosis (LSTS) and to explore the relationship of comorbidities and parental mental health with HRQoL outcomes., Methods: A cross-sectional study was undertaken with children who had undergone slide tracheoplasty. Participants included parents and children (age 5-15 years) recruited over a 13-month period, who were asked to complete validated measures of HRQoL, development and behaviour. Scores were compared to published norms., Results: Forty-two children (male 69%; n = 29) were included; mean age was 5.3 (standard deviation 3.5) years and mean follow-up was 45 (range 4-179) months. Mean total HRQoL scores for children with repaired LSTS did not differ from those of healthy norms other than for children aged 13-23 months, but 10 children (24%) had scores >2 SD below the mean for healthy children. HRQoL was poorer in children with non-cardiac congenital comorbidities than in those with isolated LSTS (mean scores 60.34 ± 17.19 and 85.52 ± 12.19, respectively, P = 0.01). There was good agreement between children's and parents' scores, although children rated their HRQoL as better than their parents did. Anxious parents rated their children's HRQoL as significantly worse than non-anxious parents (P<0.001)., Conclusions: Older children with isolated LSTS can have excellent HRQoL after surgery. Younger children, at an earlier time point postoperatively, and those with non-cardiac congenital comorbidities have poorer HRQoL. Further longitudinal evaluation is required to identify psycho-social (including parental) predictors of outcome which may inform, or be amenable to, intervention., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Recently a new in vivo approach in man, using a regional intestinal perfusion technique, has been developed. The perfusion tube consists of a multichannel tube with two inflatable balloons, which are placed 10 cm apart. The tube is introduced orally and the time required for insertion and positioning of the tube is approximately 1 hr. In the present study eight healthy subjects were perfused in the proximal jejunum on three separate occasions. The first two perfusion experiments used the same flow rate, 3 ml/min, and the third experiment used 6 ml/min. Phenazone (antipyrine) was chosen as the model drug. The recovery of PEG 4000 in the outlet intestinal perfusate was complete in experiments 1 and 2, but slightly lower (90%) when the higher flow rate was used. The mean (+/- SD) fraction of phenazone absorbed calculated from perfusion data was 51 +/- 12% (3 ml/min), 64 +/- 19% (3 ml/min), and 42 +/- 27% (6 ml/min) for the three experiments, respectively. The mean fraction absorbed estimated by deconvolution of the plasma data was 47 +/- 16%, 51 +/- 19%, and 38 +/- 26%, respectively. The effective permeability of phenazone was 5.3 +/- 2.5, 11 +/- 6.8, and 11 +/- 12 (x 10(4) cm/sec, respectively. We have shown that it was possible to establish a tight intestinal segment which behaved as a well-mixed compartment. The low perfusion rate of 3 ml/min was preferred, since it resulted in the lowest variability in absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of azodisal sodium (ADS) on the transit of a radioactive marker in the small bowel was compared with that of 5-ASA and sulfasalazine (SASP). The bile-excreted radiopharmaceutic 99mTc-HIDA was infused intravenously for 1 h into conscious rats with an intact alimentary tract and the radioactivity along the excised gastrointestinal specimen was subsequently determined. After administration of ADS (12.5 mg/rat), 5-ASA and SASP, the distribution of radioactivity was characterized by localized peaks of radioactivity separated by fairly long empty regions. As judged from experience this pattern represents the fasting state under control conditions. Administration of ADS (60 and 120 mg/rat) resulted in a distribution along the small bowel without any apparent peaks or empty regions. The transport rate of the front of the tracer was enhanced by ADS at higher doses but not by 5-ASA or SASP. Intravenous administration of ADS did not affect the transit, indicating the importance of the presence of ADS in the gut lumen. Prostaglandin release does not seem to be a probable mediating mechanism since pretreatment with indomethacin did not abolish the effect of ADS on the interdigestive transit pattern.

Azodisalicylate, a second generation drug of sulfasalazine, delivers twice the amount of 5-aminosalicylic acid to the colonic lumen on a molar basis when split by bacterial azoreductases. In 6 patients with inactive ulcerative colitis, the colonic metabolism of sulfasalazine and azodisalicylate was studied by equilibrium in vivo dialysis of feces to measure the therapeutically relevant concentrations of their metabolites (5-aminosalicylic acid and N-acetyl-5-aminosalicylic acid) in free fecal water. After oral intake of sulfasalazine (2 g/day) the total luminal concentrations of 5-aminosalicylic acid and N-acetyl-5-aminosalicylic acid determined by high-performance liquid chromatography were higher (median 14 mmol/L) than previously reported and almost doubled (median 25 mmol/L, p less than 0.05) when sulfasalazine was replaced by the same dose of azodisalicylate. In contrast, the corresponding serum concentrations were negligible. After administration of azodisalicylate to 12 healthy volunteers, a similar distribution of the metabolites was found. In nearly all cases, the concentrations of azodisalicylate in fecal dialysates were below the detection limit (6 mumol/L). We conclude, therefore, that oral azodisalicylate is a highly effective means of delivery of 5-aminosalicylic acid to the colonic mucosa. In addition, determination of the active metabolites in free fecal water seems important for the interpretation of results obtained in vitro concerning the mode of drug action.

The effect of a peroral dose of olsalazine sodium on the migrating motor complexes (MMC) in the upper small bowel was studied by manometry in nine healthy volunteers during fasting. Recordings were obtained from the duodenum and/or the upper jejunum for 4.0 - 7.5 h. During total baseline observation periods of 30 h, fourteen MMCs could be identified compared to ten complexes during post dose registrations of 25 h. This gives an average mean interval for baseline pre dose complexes of 2.2 h, and for post dose complexes 2.5 h. If excluding the two subjects, who had the longest interval until the first MMC before dosing and did not present any MMC after dosing, the corresponding figures are 1.9 h and 1.9 h, respectively. Although our material is small with respect to the large individual variation in the recordings the results indicate that MMC does occur after an olsalazine sodium dose which is twice the dose normally recommended. Besides we can conclude that there is no apparent interference with the time for MMC occurrence in the upper small bowel in fasting humans.

The biliary excretion of Olsalazine sodium (ADS) was studied by three different methods in healthy volunteers and in patients. 1 g ADS in a 2% solution was infused during 1 h into jejunum in six healthy volunteers via a three lumen sond. The bile was collected, during 2.5-6 h, proximal of an occlusive balloon on the three lumen sond. 10 mg ADS was given i.v. to five healthy volunteers and the bile was collected during 3-4.5 h with the same type of three lumen sond as used in the first experiment. 1 g ADS was given orally to three patients, with a T-tube inserted into coleducus at surgery. The bile was collected via the T-tube for 24 h. The mean biliary excretion of the jejunal dose was 0.41% (0.22% if one subject, who probably had a beckflow of the instillation fluid, is omitted). In patients, the mean excretion with bile of ADS was 0.35% and of ac-5-ASA 0.17%. Due to the short collection time after jejunal infusion and the probably incomplete collection of bile in both enteral studies the biliary excretion was estimated to be less than 2% as ADS and less than 1% as ac-5-ASA. The biliary excretion of an i.v. dose showed large individual variations (0.16-12.2%) which were not correlated to the length of the collection time. The total excretion was estimated to be less than 20% as ADS. No metabolites were detected after the i.v. dose.