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Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O 2 , airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na + and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na + channel) subunit expression. The combination of increased Na + absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

The records of the sterilization processes of 2 practices from the period 2012-2019 were analysed. The study evaluated whether sterilization processes with an additional steam penetration test gave a complete colour change. A total of 13,923 sterilization runs were evaluated. Reasons for unsuccessful sterilization runs were damp instruments (35%) or an error message on the sterilization apparatus display (35%). Of the 635 sterilization runs with the additional TST strip a complete colour change was observed in all cases. Of the 250 sterilization runs using an additional Helix Test, an incomplete colour change was observed in 2 cases. Based on this retrospective analysis, carrying out an additional test (TST strip or Helix Test) on a weekly basis did not appear to contribute to the detection of irregularities. Visual evaluation and checking the display following the sterilization process did do so.

Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell-deficient and P2X7 receptor-deficient (P2X7-/-) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell-deficient mice with WT mast cells and P2X7-/- mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7-/- mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR.

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mucociliary clearance (MCC) is a dominant component of pulmonary host defense. In health, the periciliary layer (PCL) is optimally hydrated, thus acting as an efficient lubricant layer over which the mucus layer moves by ciliary force. Airway surface dehydration and production of hyperconcentrated mucus is a common feature of chronic obstructive lung diseases such as cystic fibrosis (CF) and chronic bronchitis (CB). Mucus hydration is driven by electrolyte transport activities, which in turn are regulated by airway epithelial purinergic receptors. The activity of these receptors is controlled by the extracellular concentrations of ATP and its metabolite adenosine. Vesicular and conducted pathways contribute to ATP release from airway epithelial cells. In this study, we review the evidence leading to the identification of major components of these pathways: (a) the vesicular nucleotide transporter VNUT (the product of the SLC17A9 gene), the ATP transporter mediating ATP storage in (and release from) mucin granules and secretory vesicles; and (b) the ATP conduit pannexin 1 expressed in non-mucous airway epithelial cells. We further illustrate that ablation of pannexin 1 reduces, at least in part, airway surface liquid (ASL) volume production, ciliary beating, and MCC rates.

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl - channel controlled in part by the adenosine A 2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y 2 receptor (P2Y 2 R) that activates Ca 2+ -regulated Cl - channels (CaCC/TMEM16) and inhibits Na + absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y 2 R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ 32 P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co 4 (H 2 O) 2 (PW 9 O 34 ) 2 ] 10- (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

Background: To be fully activated platelets are dependent on two positive feedback loops; the formation of thromboxane A2 by cyclooxygenase in the platelets and the release of ADP. We wanted to evaluate the effect of aspirin on platelet function in patients with acute coronary syndromes and we hypothesized that increased levels of ADP in patients with acute coronary syndromes could contribute to aspirin resistance., Methods: Platelet activity in 135 patients admitted for chest pain was assessed with PFA-100. An epinephrine-collagen cartridge (EPI-COLL) was used for the detection of aspirin resistance together with an ADP-collagen cartridge (ADP-COLL). ADP was measured with hplc from antecubital vein samples. Three subgroups were compared: chest pain with no sign of cardiac disease (NCD), NonST-elevation myocardial infarction (NSTEMI) and STEMI., Results: Platelet activation was increased for the STEMI group compared NCD. Aspirin resistance defined as <193 sec in EPI-COLL was 9.7 % in NCD, and increased to 26.0 % (n.s.) in NSTEMI and 83.3 % (p < 0.001) in STEMI. Chronic aspirin treatment significantly reduced platelet aggregation in NCD and NSTEMI, but it had no effect in STEMI. Plasma levels of ADP were markedly increased in STEMI (905 +/- 721 nmol/l, p < 0.01), but not in NSTEMI (317 +/- 245), compared to NCD (334 +/- 271, mean +/- SD). ADP levels correlated with increased platelet activity measured with ADP-COLL (r = -0.30, p < 0.05). Aspirin resistant patients (EPI-COLL < 193 sec) had higher ADP levels compared to aspirin responders (734 +/- 807 vs. 282 +/- 187 nmol/l, mean +/- SD, p < 0.05)., Conclusion: Platelets are activated and aspirin resistance is more frequent in STEMI, probably due to a general activation of platelets. ADP levels are increased in STEMI and correlates with platelet activation. Increased levels of ADP could be one reason for increased platelet activity and aspirin resistance.

Background: Extracellular uridine triphosphate (UTP) stimulates vasodilatation, automaticity in ventricular myocytes and release of tissue-plasminogen activator (t-PA), indicating that UTP may be important in cardiac regulation. We took advantage of a recently developed quantitative assay for UTP to test the hypothesis that UTP is released in the circulation during cardiac ischemia., Methods: In ten pigs, a balloon catheter in the left anterior descending artery was introduced to induce ischemia. Samples were collected from the coronary sinus. Blood flow in the coronary sinus was assessed by a Doppler velocity transducer., Results: Plasma UTP levels increased early during ischemia and early after reperfusion (by 257+/-100 and 247+/-72%, p<0.05). Cardiac blood flow, ventricular arrhythmias and t-PA release were markedly increased at the same time points. In contrast, after 30 min, a second period of ischemia did not result in any significant increase of UTP or blood flow. Furthermore, ventricular arrhythmias were less frequent. UTP levels correlated with ventricular arrhythmia and blood flow. Similar results were found for ATP., Conclusion: For the first time we have shown that UTP is released during cardiac ischemia. UTP released during ischemia may stimulate blood flow, arrhythmia and t-PA release.

Objective: To assess interobserver variation in the diagnosis of thick tissue specimens (microbiopsies) in cytology smears and histologic sections taken from them, to evaluate the applicability of MIB-1 in histologic sections from microbiopsies and to evaluate whether processing microbiopsies in inconclusive smears has additional diagnostic value., Study Design: Cytologic smears were selected in which there were diagnostic disagreements between pathologists and cytologists and microbiopsies were present. Interobserver variation among three pathologists and three cytologists in the diagnosis of these microbiopsies was investigated. The smears were processed for histologic sections, and interobserver variation between pathologist diagnoses were analyzed. An additional histologic slide stained for MIB-1 was used for consensus diagnosis. The consensus diagnosis was compared with available follow-up and its sensitivity and specificity determined. The value of applying the microbiopsy technique in slides diagnosed as inadequate or atypical squamous cells of undetermined significance (ASCUS) was analysed., Results: From a series of 62,334 cervical smears, 49 with microbiopsies were selected. It was possible to derive histologic slides from 38 cases. Interobserver variability in the diagnosis of microbiopsies and histologic sections from them was moderate--kappa = .44 (SE = .06) and kappa = .44 (SE = .09), respectively. In the consensus meeting for all cases, a conclusive diagnosis was reached. The Pearson correlation coefficient between the consensus diagnosis and MIB-1 staining was r = .62. The sensitivity of the consensus diagnosis for the follow-up diagnosis was 71% and the specificity 60%. Diagnosis on approximately 50% of slides diagnosed as inadequate or ASCUS could be made., Conclusion: The histotechnical workup of microbiopsies is not difficult; however, their diagnosis can be a problem. Adequate diagnostic criteria are not available. Aided by MIB-1 staining, histologic sections from microbiopsies can be diagnosed, and the diagnoses correlated with follow-up in most cases. Processing of microbiopsies in smears with an inconclusive cytologic diagnosis or a diagnosis of ASCUS allowed correct diagnosis in 50% of cases in this study.

Seven analogues of the trisaccharide beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3 have been synthesized as potential substrates for glycosyltransferases involved in the chain-termination of N-acetyllactosamine-type N-glycans. These compounds include: 3-O-methyl-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp -(1-->O) (CH2)7CH3, 3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1 -->O) (CH2)7CH3, 3-deoxy-3-fluoro-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-M anp- (1-->O)(CH2)7Ch3, 3-amino-3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Ma np- (1-->O)(CH2)7CH3, beta-D-Gulp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-- >O)(CH2)7CH3, beta-L-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3, and alpha-L-Altp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1- ->O) (CH2)7CH3. All trisaccharides were obtained by condensation of suitably modified glycosyl donors based on imidates or thioglycosides with the same disaccharide acceptor, octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)-alpha-D-mannopyranoside, followed by deprotection.

Five trisaccharide derivatives designed for detailed exploration of the acceptor specificity of glycosyltransferases involved in termination of N-acetyllactosamine-type structures have been synthesized: beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->0)(CH2)7CH3 (1), 4-deoxy-beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 (2), 4-O-methyl-beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 (3), 4-deoxy-4-fluoro-beta-D-Gal p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p(1-->O)(CH2)7CH3 (4), and beta-D-Glc p-(1-->4)-beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->O)(CH2)7CH3 (5). A general disaccharide acceptor octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D -glucopyranosyl)-alpha-D-mannopyranoside was synthesized by condensation of 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-alpha, beta-D-glucopyranosyl trichloroacetimidate with octyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside, followed by deacetylation. 2,3,4,6-Tetra-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate and 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate were used as the glycosyl donors in the syntheses of 1 and 5. The modified galactosyl derivatives required subtle anomeric activation. Suitable donors for 2 turned out to be 2,3,6-tri-O-acetyl-4-deoxy-alpha-D-xylo-hexopyranosyl trichloroacetimidate and ethyl 2,3,6-tri-O-acetyl-4-deoxy-1-thio-alpha, beta-D-xylo-hexopyranoside; for 3, ethyl 2,3,6-tri-O-acetyl-4-O-methyl-1-thio-alpha, beta-D-galactopyranoside; and for 4, 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-alpha-D-galactopyranosyl trichloroacetimidate. It was concluded that thioglycosides were most appropriate for stereoselective coupling of activated synthons (carrying deoxy or O-methyl groups), whereas trichloroacetimidates gave high yields with deactivated (fluorine-containing) synthons.

Alterations in affinity of amyloid for Congo red after incubation of tissue sections with potassium permanganate, as described by Wright el al, were studied. The affinity of amyloid for Congo red after incubation with potassium permanganate did not change in patients with myeloma-associated amyloidosis, familial amyloidotic polyneuropathy, medullary carcinoma of the thyroid, pancreatic island amyloid, and cerebral amyloidosis. Affinity for Congo red was lost after incubation with potassium permanganate in tissue sections from patients with secondary amyloidosis and amyloidosis complicating familial Mediterranean fever (consisting of amyloid AA). Patients with primary amyloidosis could be divided into two groups, one with potassium-permanganate--sensitive and one with potassium-permanganate--resistant amyloid deposits. These two groups correlated with the clinical classification in typical organ distribution (presenting with nephropathy) and atypical organ distribution (presenting with cardiomyopathy, nephropathy, and glossopathy) and the expected presence of amyloid AA or amyloid AL. Potassium permanganate sensitivity seems to be restricted to amyloid AA. The potassium permanganate method can be important in dividing the major forms of generalized amyloidosis in AA amyloid and non-AA amyloid. This can be used for differentiating early stages of the disease and cases otherwise difficult to classify. It is important to define patient groups properly, especially in evaluating the effect of therapeutic measures. (Am J Pathol 97:43--58, 1979).