Postmortem Human Lungs Research Articles

SARS-CoV-2 infection dysregulates the expression of clinically relevant drug metabolizing enzymes in Vero E6 cells and membrane transporters in human lung tissues.

SARS-CoV-2-mediated interactions with drug metabolizing enzymes and membrane transporters (DMETs) in different tissues, especially lung, the main affected organ may limit the clinical efficacy and safety profile of promising COVID-19 drugs. Herein, we investigated whether SARS-CoV-2 infection could dysregulate the expression of 25 clinically relevant DMETs in Vero E6 cells and postmortem lung tissues from COVID-19 patients. Also, we assessed the role of 2 inflammatory and 4 regulatory proteins in modulating the dysregulation of DMETs in human lung tissues. We showed for the first time that SARS-CoV-2 infection dysregulates CYP3A4 and UGT1A1 at the mRNA level, as well as P-gp and MRP1 at the protein level, in Vero E6 cells and postmortem human lung tissues, respectively. We observed that at the cellular level, DMETs could potentially be dysregulated by SARS-CoV-2-associated inflammatory response and lung injury. We uncovered the pulmonary cellular localization of CYP1A2, CYP2C8, CYP2C9, and CYP2D6, as well as ENT1 and ENT2 in human lung tissues, and observed that the presence of inflammatory cells is the major driving force for the discrepancy in the localization of DMETs between COVID-19 and control human lung tissues. Because alveolar epithelial cells and lymphocytes are both sites of SARS-CoV-2 infection and localization of DMETs, we recommend further investigation of the pulmonary pharmacokinetic profile of current COVID-19 drug dosing regimen to improve clinical outcomes.

Free lung desmosine: a potential biomarker for elastic fiber injury in pulmonary emphysema

Introduction Desmosine and isodesmosine (DID) are biomarkers for elastic fibre damage in pulmonary emphysema. However, current methods for measuring lung DID involve tissue hydrolysis and lack specificity for those fibres undergoing breakdown. To address this limitation, free (nonpeptide-bound) DID content in unhydrolyzed tissues was evaluated as a more accurate biomarker in an animal model of pulmonary emphysema. Methods Hamsters were treated with either cigarette smoke and lipopolysaccharide (LPS), room air and LPS, or room air alone (controls). Free DID levels in fresh and formalin-fixed lungs were measured by LC-MS/MS and correlated with the mean linear intercept (MLI) measure of airspace size. Results There was no significant difference in free DID between fresh and formalin-fixed lungs. Animals treated with smoke and LPS had significantly higher levels of free DID than the LPS only group (359 vs. 93.1 ng/g wet lung, respectively; p = 0.0012) and room air controls (undetectable levels; p = 0.0002). There was a significant positive correlation between free DID and MLI (p < 0.0001). Conclusions The results support the hypothesis that free lung DID is a sensitive indicator of alveolar wall injury that may be used to study the development of pulmonary emphysema in both animal models and post-mortem human lung tissue.

TLR3 absence confers increased survival with improved macrophage activity against pneumonia.

Toll-like receptor 3 (TLR3) is a pathogen recognition molecule associated with viral infection with double-stranded RNA (dsRNA) as its ligand. We evaluated the role of TLR3 in bacterial pneumonia using Klebsiella pneumoniae (KP). WT and TLR3-/- mice were subjected to a lethal model of KP. Alveolar macrophage polarization, bactericidal activity, and phagocytic capacity were compared. RNA-sequencing was performed on alveolar macrophages from the WT and TLR3-/- mice. Adoptive transfers of alveolar macrophages from TLR3-/- mice to WT mice with KP were evaluated for survival. Expression of TLR3 in postmortem human lung samples from patients who died from gram-negative pneumonia and pathological grading of pneumonitis was determined. Mortality was significantly lower in TLR3-/-, and survival improved in WT mice following antibody neutralization of TLR3 and with TLR3/dsRNA complex inhibitor. Alveolar macrophages from TLR3-/- mice demonstrated increased bactericidal and phagocytic capacity. RNA-sequencing showed an increased production of chemokines in TLR3-/- mice. Adoptive transfer of alveolar macrophages from the TLR3-/- mice restored the survival in WT mice. Human lung samples demonstrated a good correlation between the grade of pneumonitis and TLR3 expression. These data represent a paradigm shift in understanding the mechanistic role of TLR3 in bacterial pneumonia.

Lung Purinoceptor Activation Triggers Ventilator-Induced Brain Injury

Objectives:Mechanical ventilation can cause ventilator-induced brain injury via afferent vagal signaling and hippocampal neurotransmitter imbalances. The triggering mechanisms for vagal signaling during mechanical ventilation are unknown. The objective of this study was to assess whether pulmonary transient receptor potential vanilloid type-4 (TRPV4) mechanoreceptors and vagal afferent purinergic receptors (P2X) act as triggers of ventilator-induced brain injury.Design:Controlled, human in vitro and ex vivo studies, as well as murine in vivo laboratory studies.Setting:Research laboratory.Subjects:Wild-type, TRPV4-deficient C57BL/6J mice, 8–10 weeks old. Human postmortem lung tissue and human lung epithelial cell line BEAS-2B.Intervention:Mice subjected to mechanical ventilation were studied using functional MRI to assess hippocampal activity. The effects of lidocaine (a nonselective ion-channel inhibitor), P2X-purinoceptor antagonist (iso-PPADS), or genetic TRPV4 deficiency on hippocampal dopamine-dependent pro-apoptotic signaling were studied in mechanically ventilated mice. Human lung epithelial cells (BEAS-2B) were used to study the effects of mechanical stretch on TRPV4 and P2X expression and activation. TRPV4 levels were measured in postmortem lung tissue from ventilated and nonventilated patients.Measurements and Main Results:Hippocampus functional MRI analysis revealed considerable changes in response to the increase in tidal volume during mechanical ventilation. Intratracheal lidocaine, iso-PPADS, and TRPV4 genetic deficiency protected mice against ventilationinduced hippocampal pro-apoptotic signaling. Mechanical stretch in both, BEAS-2B cells and ventilated wild-type mice, resulted in TRPV4 activation and reduced Trpv4 and P2x expression. Intratracheal replenishment of adenosine triphosphate in Trpv4–/– mice abrogated the protective effect of TRPV4 deficiency. Autopsy lung tissue from ventilated patients showed decreased lung TRPV4 levels compared with nonventilatedConclusions:TRPV4 mechanosensors and purinergic receptors are involved in the mechanisms of ventilator-induced brain injury. Inhibition of this neural signaling, either using nonspecific or specific inhibitors targeting the TRPV4/adenosine triphosphate/P2X signaling axis, may represent a novel strategy to prevent or treat ventilator-induced brain injury.

Fritz Rohrer (1888-1926), a pioneer in pulmonary mechanics whose work was inexplicably ignored for about 30 years.

Fritz Rohrer (1888-1926) has a special place in the history of respiratory physiology for two reasons. The first is that he laid the foundations of modern pulmonary mechanics in the early 1900s. For example, his seminal paper on pulmonary dynamics, that is, the pressure-flow relationships in the airways, was published in 1915 in one of the top journals in the field. It included extensive measurements of airway dimensions in postmortem human lungs and a sophisticated analysis of the modes of airflow. This was closely followed by a very original analysis of lung statics, which included studies of airway pressures at normal, maximal, and minimal lung volumes in relaxed normal volunteers, and was published in 1916. Remarkably, both papers were essentially ignored at the time. Fortunately, in 1925 he was able to summarize his major findings in a chapter in an important handbook of physiology. However, he tragically died from pulmonary tuberculosis in the following year at the early age of 37. The second reason for his importance in the history of pulmonary mechanics is that inexplicably his very innovative research was essentially ignored for about 30 years. It was not until the 1940s that his work was rediscovered, although not in time to save investigators from duplicating his very original studies. Possible reasons why his work was ignored for so long are discussed. Even today it is not easy to recover some important features of his career, and some aspects of his very original research are still almost unknown.

Native matrix-based human lung alveolar tissue model in vitro: studies of the reparatory actions of mesenchymal stem cells.

Studies of lung diseases in vitro often rely on flat, plastic-based monocultures, due to short lifespan of primary cells, complicated anatomy, lack of explants, etc. We hereby present a native 3D model with cues for repopulating epithelial cells. Abilities of mesenchymal stem cells (MSC) to modulate bacterial lipopolysaccharide (LPS) and cigarette smoke-induced injury to pulmonary epithelium were tested in our model. Post-mortem human lung tissue was sliced, cut and decellularized. Resulting matrix pads were reseeded with pulmonary epithelium (A549 line). Markers of the layer integrity and certain secreted proteins in the presence of cigarette smoke extract (CSE) and LPS were assessed via Western blot, ELISA and RT-PCR assays. In parallel, the effects of MSC paracrine factors on exposed epithelial cells were also investigated at gene and protein levels. When cultured on native 3D matrix, A549 cells obtain dual, type I- and II-like morphology. Exposure to CSE and LPS leads to downregulation of several epithelial proteins and suppressed proliferation rate. MSC medium added to the model restores proliferation rate and some of the epithelial proteins, i.e. e-cadherin and beta-catenin. CSE also increases secretion of pro-inflammatory cytokines by epithelial cells and upregulates transcription factor NFκB. Some of these effects might be counteracted by MSC in our model. We introduce repopulated decellularized lung matrix that highly resembles in vivo situation and is convenient for studies of disease pathogenesis, cytotoxicology and for exploring therapeutic strategies in the human lung context in vitro. MSC paracrine products have produced protecting effects in our model.

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

Determination of organochlorine pesticides and polychlorinated biphenyls in post-mortem human lung by matrix solid-phase dispersion with the aid of response surface methodology and desirability function

A matrix solid-phase dispersion (MSPD) method for the simultaneous determination of 20 organochlorine pesticides (OCPs) (aldrin, endrin, dieldrin, α-BHC, β-BHC, γ-BHC, δ-BHC, α-chlordane, γ-chlordane, p,p′-DDE, p,p′-DDT, p,p′-DDD, endosulfan I, endosulfan II, endosulfan sulfate, endrin aldehyde, heptachlor, heptachlor epoxide, endrin ketone and methoxychlor) and six polychlorinated biphenyl (PCB) congeners (PCB 28, 52, 101, 138, 153 and 180) in post-mortem human lung has been developed and validated. Response surface methodology (RSM) and desirability function were employed to optimize the extraction conditions of MSPD. Extraction was carried out using Florisil (2.0g) as the sorbent material as well as clean-up adsorbent (1.5401g), n-hexane:dichloromethane (11:89, v/v) as the eluting solvent (15.45mL) and Na2SO4 (2.0g) as dehydrating agent. Determination and quantification of OCPs and PCBs residues were carried out using a gas chromatograph equipped with an electron capture detector (GC-ECD). A mass spectrometric detector (GC–MS) in the selected ion monitoring (SIM) mode was also used for confirmation purposes. Method detection limits by GC–MS ranged from 0.42 to 0.87ngg−1 and 0.51 to 1.35ngg−1, for OCPs and PCBs, respectively. Lower detection limits were calculated for GC-ECD ranging between 0.15–0.30ngg−1 and 0.18–0.48ngg−1, respectively. Relative standard deviations did not exceed 9%. Analytes provided recoveries ranging from 65% to 106%. The proposed method was successfully applied to the analysis of lung tissues from six autopsy cases.

Induction of the Plasminogen Activator System by Mechanical Stimulation of Human Bronchial Epithelial Cells

Mechanical stimulation of the airway epithelium, as would occur during bronchoconstriction, is a potent stimulus and can activate profibrotic pathways. We used DNA microarray technology to examine gene expression in compressed normal human bronchial epithelial cells (NHBE). Compressive stress applied continuously over an 8-h period to NHBE cells led to the upregulation of several families of genes, including a family of plasminogen-related genes that were previously not known to be regulated in this system. Real-time PCR demonstrated a peak increase in gene expression of 8.0-fold for urokinase plasminogen activator (uPA), 16.2-fold for urokinase plasminogen activator receptor (uPAR), 4.2-fold for plasminogen activator inhibitor-1 (PAI-1), and 3.9-fold for tissue plasminogen activator (tPA). Compressive stress also increased uPA protein levels in the cell lysates (112.0 versus 82.0 ng/ml, P = 0.0004), and increased uPA (4.7 versus 3.3 ng/ml, P = 0.02), uPAR (1.3 versus 0.86 ng/ml, P = 0.007), and PAI-1 (50 versus 36 ng/ml, P = 0.006) protein levels in cell culture media. Functional studies demonstrated increased urokinase-dependent plasmin generation in compression-stimulated cells (0.0090 versus 0.0033 OD/min, P = 0.03). In addition, compression led to increased activation of matrix metalloproteinase (MMP)-9 and MMP-2 in a urokinase-dependent manner. In postmortem human lung tissue, we observed an increase in epithelial uPA and uPAR immunostaining in the airways of two patients who died in status asthmaticus compared with minimal immunoreactivity noted in airways from seven lung donors without asthma. Together these observations suggest an integrated response of airway epithelial cells to mechanical stimulation, acting through the plasminogen-activating system to modify the airway microenvironment.

Bioelectric properties of cultured monolayers from epithelium of distal human fetal lung

Throughout intrauterine life, Cl(-)-rich liquid is secreted by the pulmonary epithelium. To evaluate the role of the most distal epithelium in liquid secretion, we measured bioelectric properties of monolayers composed of epithelial cells from acinar structures of postmortem human fetal lung (mean gestation, 22.3 wk; range, 18-24 wk). These monolayers formed high-resistance (R) barriers (mean R = 363 Ohm/cm2) when cultured in hormone-supplemented, serum-free medium. The transepithelial electrical potential difference (4.0 mV, lumen negative), was similar to that of whole fetal sheep lung in vivo. Equivalent short-circuit current (Ieq) was inhibited by apical amiloride (-20%), 5-(N-ethyl-N-isopropyl)-amiloride (-33 to -49%), or diphenylamine-2-carboxylate (DPC; -26%), and by basolateral ouabain (-77%), whereas apical 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) had no effect. Bumetanide added to the basolateral bath did not affect resting Ieq, but inhibited Ieq (-19%) in monolayers pretreated with apical amiloride, basolateral terbutaline, and apical ATP, and also inhibited Ieq (-22%) of monolayers pretreated with basolateral amiloride and DIDS. Ieq was stimulated by terbutaline (90-128%), ATP (70-186%), and ionomycin (141%). Stimulation of Ieq by these agents is compatible with induction of Cl- secretion through two pathways: channels that are opened by a rise in adenosine 3',5'-cyclic monophosphate, and channels that are opened by a rise in intracellular Ca2+. Inhibition of Ieq by apical DPC implies that Cl- secretion may contribute to basal Ieq.

Adsorption of fetal surfactant protein SP-B on the human amnion at term and on amniocytes incubated with fetal surfactant in vitro

Fetal surfactant stimulates the synthesis of prostaglandins by slices of human amnion at term and by a human amnion cell line, and these effects are partly dependent upon surfactant apoproteins. In this paper, methods are described for the purification of surfactant from human amniotic fluid and from post-mortem human lung. A procedure is described for the purification of surfactant protein SP-B from human amniotic fluid, and the sequence of 20 amino acids at the N-terminal has been determined. A monoclonal antibody generated against human lung surfactant has been shown to react with SP-B from amniotic-fluid surfactant, and the presence of SP-B on the surface of the amnion at term has been demonstrated by immunohistochemical methods. It has also been shown that SP-B from surfactant is present on the surface of amniocytes incubated with surfactant in vitro.

The Utility of Postmortem Lung for RNA Studies; Variability and Correlation of the Expression of Surfactant Proteins in Human Lung

Postmortem human lung tissue was evaluated for its utility in studies of the mRNAs for the surfactant proteins. Data obtained from different analytical procedures indicated that surfactant protein mRNAs are quite stable in these tissues with a half-life of 10 to 12 h. These analyses revealed no major regional differences in the mRNA levels for the surfactant protein A (SP-A) and surfactant protein B (SP-B) although small differences were present in the levels for the surfactant protein C (SP-C). Analysis of adult surgical lung specimens indicated that there is greater individual variation in the mRNA levels for SP-A and SP-B compared to SP-C among individuals. Furthermore, in a given individual the level of SP-A mRNA correlated well with that of SP-B, whereas the level of SP-C mRNA did not correlate with either that of SP-A or SP-B.

Elasticity of arterioles and venules in postmortem human lungs

The elasticity and branching order of noncapillary microscopic blood vessels less than 100 microns diam were studied in human lungs obtained 7-30 h postmortem, using a silicone elastomer method that selectively filled pulmonary arterioles or venules. The lungs were inflated to 10 cmH2O pressure and a gradient of transmural vascular pressure of 0-17 cm H2O, from lobe base to apex, was established in the silicone-filled vascular system. Histological materials were obtained after airway fixation by formaldehyde solution and analyzed for vessel diameter in the branching order of 1, 2, and 3, with the smallest noncapillary vessel designated as order 1, in accord with the Strahler system. The change in vessel diameter within a branching order at different levels of transmural pressure is a derived measure of vascular elasticity expressed as compliance coefficient alpha, alpha Values are 0.128, 0.164, and 0.210 micron/cmH2O or 0.682, 0.472, and 0.354%/cmH2O, respectively, of orders 1-3 for arterioles and 0.187, 0.215, and 0.250 micron/cmH2O or 0.992, 0.612, and 0.424%/cmH2O, respectively, of orders 1-3 for venules. The percent is normalized with D0, which is the value of diameter (D) when the transmural pressure is zero. These data are compared with those for the cat where alpha = 0.274 for similar juxta-alveolar vessels.

Neurotensin in human small cell lung carcinoma.

High levels of neurotensin-like immunoreactivity were found in human small cell lung carcinoma lines. No immunoreactivity was present in non-small cell carcinoma lines and only low amounts in postmortem human lung tissue. The immunoreactive material co-eluted with synthetic neurotensin on two different chromatographic systems. No evidence was obtained for the presence of specific neurotensin binding sites in any of the small cell carcinoma lines examined. The results suggest that small lung cell carcinoma lines may be useful for studying the biosynthesis of human neurotensin.

Peripheral airways as a determinant of ventilatory function in the human lung.

We have investigated the morphological differences responsible for the variability in two tests of pulmonary function, maximal expiratory flow rates (MEF) and the frequency dependence of dynamic compliance (CDYN ratio). Functional measurements were obtained from 53 normal and minimally diseased postmortem human lungs. Morphological measurements performed on these same lungs included airway diameter at three levels in the bronchial tree, the amount of bronchial gland mass, and the alveolar surface to volume ratio. Multiple regression analysis suggests that the diameter of the peripheral conduction airways (membranous bronchioles) is the major morphological determinant for both MEF and the CDYN ratio in lungs at any particular age. Age-dependent changes in both functional tests were associated primarily with differences in the alveolar surface to volume ratio. Minimal emphysema and a lesion associated with cigarette smoking, respiratory bronchiolitis, have no demonstrable effect on either MEF or the CDYN ratio. These studies provide further evidence that the peripheral conducting airways are a major determinant of ventilatory function in the normal human lung.

Elastic behavior of postmortem human lungs: effects of aging and mild emphysema.

The elastic behavior of postmortem human lungs has been studied in an effort to differentiate the effects of normal aging from those of mild emphysema. Static pressure-volume (P-V) curves were measured in 50 lungs obtained from men 15-85 yr of age, including 12 lungs with mild-to-moderate emphysema. The emphysema was quantitatively assessed by gross and microscopic methods. The P-V relationship in all lungs is accurately described by the empirically fitted equation, P = alpha1ea2v. This expression is useful because the two parameters separate the effects of elastic behavior (alpha1) from size (alpha2) on the P-V curve. There is a close negative correlation (R = -0.94) Between age and alpha1 in normal lungs but no significant age dependence of alpha2. Further decreases in alpha1 are found in most emphysematous lungs. Alpha1 is more than 2 SEE below the age-predicted mean in five of nine lungs with minimal emphysema (1-10% by point count) and more than 5 SEE below the mean in the three more severely affected lungs. There is a close correlation (R = +0.90) between alpha1 and the alveolar surface-to-volume ratio in both normal and emphysematous lungs.

Ultrasonic pulmonary Densitometry: preliminary studies.

The feasibility of using A-mode ultrasound to measure lung density changes was tested in two studies. (A) Wet sponges were used as experimental analogs of the lung. The sponges were weighed. A-mode recordings were obtained by placing the transducer face against a sheet of film in contact with each sponge. Ultrasound penetration increased directly with increasing water content. The range of densities studied corresponds with that which occurs in normal and edematous human lungs. In a second study (B), the depth of ultrasound penetration into 3 postmortem human lungs was measured at increasing levels of inflation by positive intrabronchial pressure. Ultrasound penetration decreased approximately logarithmically with increasing inflation pressure. These relationships indicate a potential application of ultrasound to pulmonary densitometry. This could provide a noninvasive means for clinical estimation of changes in lung water, without the use of ionizing radiation.

Basic Pattern of Tissue Repair in Human Lungs Following Unspecific Injury

A method is described by which tissue samples from postmortem human lungs may be adequately fixed for electron microscopy. An electron microscopy and morphometry study of lungs of patients dying of adult respiratory distress syndrome revealed that the alveolar epithelium was transformed to a vastly cuboidal epithelium irrespective of the inspired oxygen concentration, which in several cases did not exceed 40 percent. This constant change in alveolar epithelium, resulting in a consistent overall thickening of the epithelial barrier by a factor of 4:5 in all cases, was interpreted to represent a characteristic reaction pattern of alveolar epithelium to various kinds of injury. Arguments are presented to indicate that hypertrophy of cuboidal type 2 epithelial cells is a necessary step in the repair process, since type 1 cells are unable to divide by mitosis.

Reduction in Peripheral Airway Resistance in Excised Emphysematous Lungs

Bronchiolar mucus is a common autopsy finding in patients who die with chronic airway obstruction. Hogg et al 1 Hogg JC Macklem PT Thurlbeck WM Site and nature of airway obstruction in chronic obstructive lung disease. New Eng J Med. 1968; 278: 1355-1360 Crossref PubMed Scopus (943) Google Scholar have shown that bronchiolar mucus and mucus plugging in emphysematous lungs is associated with increased small airway resistance and therefore with potential reversibility. We have attempted to determine how reversible such obstruction may be by measuring forced expiratory volume in one second (FEV1), expiratory flow and airway resistance in excised postmortem human lungs before and after treating them with a mucolytic agent, acetylcysteine.

The Functional and Bronchographic Evaluation of Postmortem Human Lungs: Clinical, Physiologic, Roentgenologic and Pathologic Correlations in Normal Subjects and Patients with Emphysema and Chronic Bronchitis

The Functional and Bronchographic Evaluation of Postmortem Human Lungs: Clinical, Physiologic, Roentgenologic and Pathologic Correlations in Normal Subjects and Patients with Emphysema and Chronic Bronchitis