Lamellar Bodies Research Articles

HucMSCs-derived Exosomes Promote Lung Development in Premature Birth via Wnt5a/ROCK1 Axis.

Bronchopulmonary dysplasia (BPD) frequently affects extremely preterm and low birth weight infants, with current treatments lacking specificity. Enhancing extra-uterine preterm alveoli development and repairing damage are crucial for BPD management. Here we show that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-Exos) can enhance fetal lung development in mice by delivering specific contents. Briefly, hucMSCs-Exos were extracted using ultracentrifugation and identified by transmission electron microscopy (TEM), flow cytometry, Western blot (WB), and nanoparticle tracking analysis (NTA). These exosomes were then administered to pregnant mice via tail vein injection. Embryonic lung tissues were collected at E13.5 and E18.5 via cesarean section and analyzed using hematoxylin-eosin (HE) staining, immunofluorescence, and TEM. Proteomic analysis was conducted to identify protein components in the exosomes, and WB was used to assess protein expression changes. hucMSCs-Exos from full-term infants were more effective in promoting cell proliferation than those from preterm infants. In vivo, full-term hucMSCs-Exos significantly enhanced alveolarization in fetal lung tissues. Proteomic analysis revealed higher Wnt5a expression in full-term hucMSCs-Exos, and further experiments confirmed a direct interaction between Wnt5a and ROCK1. WB also showed increased expression of the autophagy marker LC3B in the lung tissues of mice treated with full-term exosomes. In conclusion, term hucMSCs-Exos may directly regulate the phosphorylation of ROCK1 in mouse lung tissue through naturally enriched Wnt5a, thus promoting autophagy of AT2 cells and lamellar body development, and ultimately enhance the alveolarization and reducing the incidence of BPD in premature infants.

Ultrastructural analysis of lamellar bodies in type II alveolar epithelial cells in the human lung.

Pulmonary surfactant is produced by type II alveolar epithelial cells (AEC2) and stored in lamellar bodies (LBs) prior to secretion. Here, we characterize AEC2 and their LBs in the human lung ultrastructurally and quantitatively. Five human lungs were analyzed by transmission electron microscopy, serial section electron tomography and stereology. A human lung contained about 24 billion AEC2 with a mean size of about 650 µm³. The number of AEC2 as well as the total volume of LBs per lung, about 1.9 mL, strongly correlated with total lung volume. A single AEC2 contained an LB volume of about 74 µm³. This amount was packed in about 324 LBs with a mean size of 0.24 µm³. Three morphologically distinct subpopulations of LBs were identified: 1.) isolated LBs which make up the majority (average 300 per AEC2), 2.) LBs connected to each other via pores (average 23 per AEC2), and 3.) LBs connected to the plasma membrane via a fusion pore (average 1 per AEC2). Along this sequence of subpopulations, the mean size of LBs increased. LBs that are connected either with each other or to the plasma membrane contained about 14% of an AEC2´s LB volume. This is in line with the concept of an intermediate surfactant pool, stored in LBs either directly or indirectly connected to the plasma membrane. In summary, this study provides quantitative reference data on surfactant-storing LBs in AEC2 as well as morphological evidence for an intermediate surfactant pool in the human lung.

Lamellar Body Count (LBC) in amniotic fluid for prediction of fetal lung maturity

Background: Neonatal respiratory distress syndrome (RDS) is a disorder due to pulmonary immaturity with a high mortality characterized by low levels of pulmonary surfactant. RDS is one of the important causes of mortality in neonates. Objective: The aim of the study is to determine the lamellar body count (LBC) cutoff value for foetal lung maturity and to evaluate the clinical usefulness of LBC in predicting the neonatal respiratory distress syndrome (RDS) and to find if the Measurement of LBC can replace the conventional Lecithin/Sphingomyelin ratio. Methods: This prospective observational study was performed on total 47 pregnant women divided as 19 patients with premature labor and 28 patients with healthy full-term singleton pregnancy as controls, with gestational age between 28 – 41 weeks at Ain Shams university maternity hospital in period between April 2014 and January 2015 with inclusion and exclusion criteria. Results: RDS was diagnosed in 20 neonates, two neonates in the 1st group (7.1%) While 18 neonates in 2nd (94.7%) showed development of RDS. Mean lamellar body count in newborns with RDS: 39,200 ± 12,438 with median 37,500 and p-value was <0.0001. ROC (receiver operator characteristics) curve showed that by using 51000/µL as a cut-off point for LBC it is a good predictor for fetal lung maturity with sensitivity 95% and specificity 96.3%. There is a direct relationship between the gestational age and the lamellar body count as the lamellar body count increases as long as the gestational age proceeds with a highly significant positive correlation (p value <0.0001). Conclusion: As evident from the current study, LBC count now replacing the conventional Lecithin/Sphingomyelin ratio because the test can be performed with equipment found in most clinical analysis laboratories and is reliable in predicting foetal lung maturity. This study suggests that LBC cutoff value of ≥51,000/µL can predict pulmonary maturity and reduce the risk of neonatal respiratory distress syndrome.

Role of Mesenchymal Stem Cells Exosomes in Lipopolysaccharide-induced Acute Lung Injury in Rats, A Histological Study

Abstract Background Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a substantial disease burden in terms of both morbidity and mortality. And since 2019 this condition represented the leading cause of mortality in patients with severe COVID-19. Mesenchymal stem cells (MSCs) and their exosomes have shown promising results in the modulation of inflammatory responses. Exosomes, have been offering a powerful cell-free therapeutic modality, bypassing most of the safety concerns related to cell-based therapy. Lipopolysaccharide (LPS), a glycoprotein present in the cell wall of gram negative bacteria, could induce ALI in experimental animals, mimicking ARDS in humans. Aim Histological assessment of the potential role of exosomes derived from bone marrow MSCs in treatment of LPS-induced acute lung injury model in adult male albino rats. Materials & Methods Thirty-five rats were used including 5 young aged albino rats that were sacrificed to harvest their bone marrow-derived MSCs and their exosomes. The remaining 30 adult male albino rats were divided into 3 groups. Group I (control group), Group II (LPS group): received a single intraperitoneal injection of LPS dissolved in PBS (6mg/kg). Group III (LPS+Exosomes group): received LPS as in group II followed by intravenous injection of Exosomes (30uL/rat), one hour later. Twenty-four hours later, lung specimens were processed for light & electron microscopic examination, as well as morphometric and statistical analysis. Results H&E-stained sections of Group II revealed marked inflammatory changes as interalveolar septa were significantly thickened by infiltrating inflammatory cells and extravasated red blood cells. Many alveoli appeared narrowed and collapsed while other alveolar spaces were dilated with destruction of the alveolar walls. Blood vessels were congested with thickened walls. The lining epithelium of bronchioles appeared occasionally desquamated and necrotic. Masson’s Trichrome-stained sections showed an apparent increase of collagen fibers around the bronchioles and in the interalveolar septa. Immunohistochemical stain showed a significantly increased reaction to iNOS antibodies in addition to a significantly increased Caspase-3 immune reactivity. Examination with Transmission electron microscopy showed Type I Pneumocyte having an electron dense nucleus and cytoplasmic vacuolations. Type II pneumocytes had degenerated lamellar bodies, many lysosomes and lost microvilli. Eosinophils were seen infiltrating the interstitium with their bilobed nucleus and specific granules with crystalline cores in addition to macrophages with large eccentric nucleus and many lysosomes. On the other hand, the lung structure in Group III showed significant improvement in all parameters. Conclusion The study showed that MSCs exosomes could help in treatment of ALI caused by LPS in rats. Group II showed signs of inflammation and lung damage in addition to increased inflammatory and apoptotic immune reactivity. However, in Group III, treatment with exosomes resulted in significant improvement in all previous parameters. This study highlights the potential of using mesenchymal stem cell exosomes as a treatment for ALI in animals and as a clinical trial.

Postnatal hypoxic preconditioning attenuates lung damage from hyperoxia in newborn mice.

Preterm infants frequently require oxygen supplementation at birth. However, preterm lung is especially sensible to structural and functional damage caused by oxygen free radicals. The adaptive mechanisms implied in the fetal-neonatal transition from a lower to a higher oxygen environment were evaluated in a murine model using a custom-designed oxy-chamber. Pregnant mice were randomly assigned to deliver in 14% (hypoxic preconditioning group) or 21% (normoxic group) oxygen environment. Eight hours after birth FiO2 was increased to 100% for 60 min and then switched to 21% in both groups. A control group remained in 21% oxygen throughout the study. Mice in the normoxic group exhibited thinning of the alveolar septa, increased cell death, increased vascular damage, and decreased synthesis of pulmonary surfactant. However, lung histology, lamellar bodies microstructure, and surfactant integrity were preserved in the hypoxic preconditioning group after the hyperoxic insult. Postnatal hyperoxia has detrimental effects on lung structure and function when preceded by normoxia compared to controls. However, postnatal hypoxic preconditioning mitigates lung damage caused by a hyperoxic insult. Hypoxic preconditioning, implemented shortly after birth mitigates lung damage caused by postnatal supplemental oxygenation. The study introduces an experimental mice model to investigate the effects of hypoxic preconditioning and its effects on lung development. This model enables researchers to delve into the intricate processes involved in postnatal lung maturation. Our findings suggest that hypoxic preconditioning may reduce lung parenchymal damage and increase pulmonary surfactant synthesis in reoxygenation strategies during postnatal care.

Establishment of Nano-ITO induced rat model of indium lung disease and exploration of its pathological characteristics

Objective: To study the dynamic pathological characteristics of lung tissue in a Nano-ITO induced rat model of indium lung disease and to guide clinical and basic scientific research to further explore the mechanisms of pulmonary interstitial injury and pulmonary alveolar proteinosis (PAP). Methods: Dose-response (three divided doses) and time-course studies (six exposure periods) were performed to investigate the pulmonary toxicity induced by Nano-ITO. At the end of the experiment, cytokine levels and oxidative stress were analyzed in the bronchoalveolar lavage fluid. Rat lung tissues were also collected for staining with H&E, PAS, Masson's, Oil Red O, and Sirius Red. Ultrastructure of lung tissue cells was observed by transmission electron microscopy. Expression of IL-1β, HO-1, SP-A was observed by immunohistochemistry, and the expression of α-SMA was observed by immunofluorescence. Results: Nano-ITO intratracheal instillation caused pulmonary toxicity by inducing acute inflammation at 3 days, granuloma (nodule) formation and collagen hyperplasia at 14 days, and alveolar proteinosis at 56 days post-exposure. Pathological features of lung tissue included typical alveolar exudates, cellular fibrous nodules, enlarged alveolar fat droplet fusion, cholesterol crystal granuloma and pulmonary alveolar proteinosis. The intra-alveolar eosinophilic material (multilamellated, lattice-shaped, and myelin-like structure) showed abnormal lamellar bodies (features of alveolar type Ⅱ epithelial cells) and abundant rough endoplasmic reticulum and mitochondria (features of fibroblasts) on transmission electron microscopy of the lung tissue from rats exposed to Nano-ITO on the 84th day. Cellular pathology revealed that a large amount of amorphous PAS stain-positive substances appear in BALF at 28 days post-exposure, and pink granular protein-like substances can be seen in alveolar macrophages. Conclusions: There are three characteristic developmental stages in Nano-ITO induced pulmonary injury in rats, acute inflammation, granuloma (nodule) formation and collagen proliferation, and pulmonary alveolar proteinosis, which provide a reference feature model for the pathogenesis of indium lung disease.

Topically applied, fatty acid-containing formulations provide superior barrier benefits in an exvivo tape-stripped skin model.

Ex vivo skin has been used to study various skin conditions from atopic dermatitis to burn injury. The aim of this research is to identify a more effective barrier improvement strategy and to evaluate topical formulations in replenishing the skin. The skin can create new longer chain fatty acids and ceramides (CERs) from topically applied skin natural fatty acid to help renew the skin's barrier. An exvivo skin model damaged by sequential tape stripping of the stratum corneum (SC) was used to investigate the repair of the SC. Confocal laser scanning microscopy was used to assess the SC layers recovered. Ultrastructural analysis was performed using transmission electron microscopy to visualize the lamellar bodies and intercellular lipid lamellae. The data in this study provide the first direct exvivo evidence comparing different marketed formulations containing three CERs with those containing fatty acids. Free fatty acid (FFA)-containing formulations, but not CER-containing formulations, directly applied to the damaged skin, showed an increased number of repaired SC layers and this was reflected at the ultrastructural level by an increased intercellular lipid lamellae length and an increased number of lamellar bodies. These findings demonstrate that FFA-containing formulations can repair damaged exvivo skin and point to a repair mechanism in which topically applied palmitic and stearic acids, (which boost lipid levels and elongation) can increase the production and transport of lipids into a repaired SC and thus rebuild an effective skin barrier.

Endogenous LXR signaling controls pulmonary surfactant homeostasis and prevents lung inflammation

Lung type 2 pneumocytes (T2Ps) and alveolar macrophages (AMs) play crucial roles in the synthesis, recycling and catabolism of surfactant material, a lipid/protein fluid essential for respiratory function. The liver X receptors (LXR), LXRα and LXRβ, are transcription factors important for lipid metabolism and inflammation. While LXR activation exerts anti-inflammatory actions in lung injury caused by lipopolysaccharide (LPS) and other inflammatory stimuli, the full extent of the endogenous LXR transcriptional activity in pulmonary homeostasis is incompletely understood. Here, using mice lacking LXRα and LXRβ as experimental models, we describe how the loss of LXRs causes pulmonary lipidosis, pulmonary congestion, fibrosis and chronic inflammation due to defective de novo synthesis and recycling of surfactant material by T2Ps and defective phagocytosis and degradation of excess surfactant by AMs. LXR-deficient T2Ps display aberrant lamellar bodies and decreased expression of genes encoding for surfactant proteins and enzymes involved in cholesterol, fatty acids, and phospholipid metabolism. Moreover, LXR-deficient lungs accumulate foamy AMs with aberrant expression of cholesterol and phospholipid metabolism genes. Using a house dust mite aeroallergen-induced mouse model of asthma, we show that LXR-deficient mice exhibit a more pronounced airway reactivity to a methacholine challenge and greater pulmonary infiltration, indicating an altered physiology of LXR-deficient lungs. Moreover, pretreatment with LXR agonists ameliorated the airway reactivity in WT mice sensitized to house dust mite extracts, confirming that LXR plays an important role in lung physiology and suggesting that agonist pharmacology could be used to treat inflammatory lung diseases.Graphical

The formation of lamellar body-like structures may be a trigger of cetylpyridinium chloride-induced cell death and inflammatory response

Cetylpyridinium chloride (CPC) is a quaternary ammonium compound used widely in health and personal care products. Meanwhile, due to its increasing use, its potential adverse health effects are emerging as a topic of public concern. In this study, we first administered CPC by pharyngeal aspiration to determine the survival level (the maximum concentration at which no death is observed) and then administered CPC to mice repeatedly for 28 days using the survival level as the highest concentration. CPC increased the total number of pulmonary cells secreting pro- and anti-inflammatory cytokines and chemokines. Infiltration of inflammatory cells, production of foamy alveolar macrophages, and chronic inflammatory lesions were found in the lung tissue of male and female mice exposed to the highest dose of CPC. We also investigated the toxicity mechanism using BEAS-2B cells isolated from normal human bronchial epithelium. At 6 h after exposure to CPC, the cells underwent non-apoptotic cell death, especially at concentrations greater than 2 μg/mL. The expression of the transferrin receptor was remarkably enhanced, and the expression of proteins that contribute to intracellular iron storage was inhibited. The expression of both mitochondrial SOD and catalase increased with CPC concentration, and PARP protein was cleaved, suggesting possible DNA damage. In addition, the internal structure of mitochondria was disrupted, and fusion between damaged organelles was observed in the cytoplasm. Most importantly, lamellar body-like structures and autophagosome-like vacuoles were found in CPC-treated cells, with enhanced expression of ABCA3 protein, a marker for lamellar body, and a docking score between ABCA3 protein and CPC was considered to be approximately −6.8969 kcal/mol. From these results, we propose that mitochondrial damage and iron depletion may contribute to CPC-induced non-apoptotic cell death and that pulmonary accumulation of cell debris may be closely associated with the inflammatory response. Furthermore, we hypothesize that the formation of lamellar body-like structures may be a trigger for CPC-induced cell death.

Overstretching alveolar epithelial type II cells decreases surfactant secretion via actin polymerization and intracellular trafficking alteration

Pulmonary surfactant is essential for maintaining proper lung function. Alveolar epithelial type II (AE2) cells secrete surfactants via lamellar bodies (LBs). In tidal loading during each breath, the physiological cyclic stretching of AE2 cells promotes surfactant secretion. Excessive stretching inhibits surfactant secretion, which is considered to contribute to the development of lung damage. However, its precise mechanism remains unknown. This study tested whether actin polymerization and intracellular transport are required for pulmonary surfactant secretion and the association of actin polymerization and transport in identical human AE2-derived A549 cells using live-cell imaging, not in the bulk cells population. We found that overstretching approximately doubled actin polymerization into filaments (F-actin) and suppressed LB secretion by half in the fluorescent area ratio, compared with physiological stretching (F-actin: 1.495 vs 0.643 (P < 0.01); LB: 0.739 vs 0.332 (P < 0.01)). An inhibitor of actin polymerization increased LB secretion. Intracellular tracking using fluorescent particles revealed that cyclic stretching shifted the particle motion perpendicularly to the direction of stretching according to the orientation of the F-actin (proportion of perpendicular axis motion prior particle: 0h 40.12 % vs 2h 63.13 % (P < 0.01)), and particle motion was restricted over time in the cells subjected to overstretching, indicating that overstretching regulates intracellular transport dynamics (proportion of stop motion particle: 0h 1.01 % vs 2h 11.04 % (P < 0.01)). These findings suggest that overstretching changes secretion through the cytoskeleton: overstretching AE2 cells inhibits pulmonary surfactant secretion, at least through accelerating actin polymerization and decreasing intracellular trafficking, and the change in actin orientation would modulate intracellular trafficking.

HPS6 Deficiency Leads to Reduced Vacuolar-Type H+-ATPase and Impaired Biogenesis of Lamellar Bodies in Alveolar Type II Cells.

Lamellar bodies (LBs) are tissue-specific lysosome-related organelles in type II alveolar cells that are the main site for the synthesis, storage, and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to their function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to a reduction of the steady-state concentration of vacuolar-type H+-ATPase and an increase in the luminal pH of LBs. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and BAL fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of vacuolar-type H+-ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.

Zebra Bodies in the Kidney: Is it a Pathognomonic Finding of Fabry Disease?

Zebra bodies are intralysosomal lamellar inclusion bodies. It is accepted as a specific feature of Fabry disease. However, it has been reported in many hereditary and acquired conditions. We are reporting Zebra bodies in the kidneys of cases with Rheumatoid Arthritis and hydroxychloroquine-induced phospholipidosis. Case 1: A 55-year-old male presented with hypertension and renal dysfunction. Serum ANA and anti-CCP antibodies were positive. A kidney biopsy revealed chronic tubulointerstitial nephritis with Zebra Bodies in the podocytes. Genetic analysis was negative for Fabry disease. Case 2: A 34-year-old female with Systemic Lupus Erythematosus on Hydroxychloroquine for a year presented with subnephrotic proteinuria. Serum ANA and anti-dsDNA antibodies were positive. Electron microscopy showed lamellated osmiophilic inclusion bodies in the tubular and visceral epithelial cells. Thus, Zebra bodies are not pathognomonic for Fabry disease and Rheumatoid Arthritis should also be considered in the differential diagnosis, particularly if family or drug history is negative.

P10 Janus kinase inhibitors to restore skin barrier function in a mouse model of Harlequin ichthyosis

Abstract Introduction and aims The autosomal recessive congenital ichthyoses (ARCIs) are a group of chronic conditions where there is unmet therapeutic need. Harlequin ichthyosis (HI) is the most severe form of ARCI with aberrant differentiation of the epidermis leading to a severe barrier defect. It is caused by mutations in ABCA12, a lipid transporter involved in transport of glucosylceramides from the lamellar body to the lipid lamellae. Several Janus kinase (JAK) inhibitors inhibiting the JAK-signal transducer and activator of transcription pathway are now licensed for atopic dermatitis. Previous work from our group in an HI in vitro model showed that tofacitinib, a pan-JAK inhibitor, had a restorative effect on the lipid barrier. Methods Further selective JAKis were tested in three dimensional skin equivalents using ABCA12 CRISPR-Cas9 knockout and wildtype keratinocytes to identify the most promising JAKi for use in an HI murine model (ABCA12tm1c: Het Cre+). JAKi-A (5 mg kg−1) and vehicle (10% dimethylsulfoxide in corn oil) were tested on male and female HI-inducible and wildtype mice (N = 4). All mice were initially treated with 4-OH-Tamoxifen to induce the HI phenotype and after 1 day JAKi-A was administered via oral gavage for 9 days. All mice were killed, and skin was collected and processed for haematoxylin and eosin, Nile Red, ABCA12, glucosylceramide and immune cell marker staining. Results JAKi-A had a positive effect on HI epidermal morphology, showing a significantly reduced epidermal thickness in treated HI mice compared with controls. JAKi-A also had a positive effect on the epidermal barrier, restoring neutral and polar lipids compared with vehicle-treated mice as observed using Nile Red and glucosylceramide staining. Further analysis on immune cell infiltrates is in progress. Conclusions These Results suggest that JAKi-A restores the skin barrier in vivo and may be a promising treatment for severe ichthyosis.

Cross-Sectional Study on Autosomal Recessive Congenital Ichthyoses: Association of Genotype with Disease Severity, Phenotypic, and Ultrastructural Features in 74 Italian Patients

Background: Autosomal recessive congenital ichthyoses (ARCIs) are a clinically heterogeneous group of keratinization disorders characterized by generalized skin scaling due to mutations in at least 12 genes. The aim of our study was to assess disease severity, phenotypic, and ultrastructural features and to evaluate their association with genetic findings in ARCI patients. Methods: Clinical signs and symptoms, and disease severity were scored in a single-center series of patients with a genetic diagnosis of ARCI. Skin ultrastructural findings were reviewed. Results: Seventy-four consecutive patients (mean age 11.0 years, range 0.1–48.8) affected with lamellar ichthyosis (50/74, 67.5%), congenital ichthyosiform erythroderma (18/74, 24.3%), harlequin ichthyosis (two/74, 2.7%), and other minor ARCI subtypes (four/74, 5.4%) were enrolled. Mutated genes were as follows: TGM1 in 18/74 (24.3%) patients, ALOX12B in 18/74 (24.3%), CYP4F22 in 12/74 (16.2%), ABCA12 in nine/74 (12.2%), ALOXE3 in seven/74 (9.5%), NIPAL4 in seven/74 (9.5%), and CERS3, PNPLA1, and SDR9C7 in 1 patient each (1.4%). Twenty-five previously undescribed mutations in the different ARCI causative genes, as well as two microduplications in TGM1, and two microdeletions in CYP4F22 and NIPAL4 were identified. The mean ichthyosis severity score in TGM1- and ABCA12-mutated patients was significantly higher than in all other mutated genes, while the lowest score was observed in CYP4F22-mutated patients. Alopecia, ectropion, and eclabium were significantly associated with TGM1 and ABCA12 mutations, and large, thick, and brownish scales with TGM1 mutations. Among specific phenotypic features, psoriasis-like lesions as well as a trunk reticulate scale pattern and striated keratoderma were present in NIPAL4-mutated patients. Ultrastructural data available for 56 patients showed a 100% specificity of cholesterol clefts for TGM1-mutated cases and revealed abnormal lamellar bodies in SDR9C7 and CERS3 patients. Conclusion: Our study expands the phenotypic and genetic characterization of ARCI by the description of statistically significant associations between disease severity, specific clinical signs, and different mutated genes. Finally, we highlighted the presence of psoriasis-like lesions in NIPAL4-ARCI patients as a novel phenotypic feature with diagnostic and possible therapeutic implications.

Mechanosensitive channels TMEM63A and TMEM63B mediate lung inflation-induced surfactant secretion.

Pulmonary surfactant is a lipoprotein complex lining the alveolar surface to decrease the surface tension and facilitate inspiration. Surfactant deficiency is often seen in premature infants and in children and adults with respiratory distress syndrome. Mechanical stretch of alveolar type 2 epithelial (AT2) cells during lung expansion is the primary physiological factor that stimulates surfactant secretion; however, it is unclear whether there is a mechanosensor dedicated to this process. Here, we show that loss of the mechanosensitive channels TMEM63A and TMEM63B (TMEM63A/B) resulted in atelectasis and respiratory failure in mice due to a deficit of surfactant secretion. TMEM63A/B were predominantly localized at the limiting membrane of the lamellar body (LB), a lysosome-related organelle that stores pulmonary surfactant and ATP in AT2 cells. Activation of TMEM63A/B channels during cell stretch facilitated the release of surfactant and ATP from LBs fused with the plasma membrane. The released ATP evoked Ca2+ signaling in AT2 cells and potentiated exocytic fusion of more LBs. Our study uncovered a vital physiological function of TMEM63 mechanosensitive channels in preparing the lungs for the first breath at birth and maintaining respiration throughout life.

Height Restoration for Vertebra Plana Using a Mechanical Lamellar Vertebral Body Reduction Device: 2-Dimensional Operative Video.

Compression fractures of thoracolumbar vertebra are the most common vertebral fracture.1 Associated with osteoporosis, the compression can progress until reach vertebra plana and cause a kyphosis.2 The reduction of the fresh fracture and restoration of the height are the purposes of the kyphoplasty.3 We present a modified technique adapted for the treatment of vertebral plana using a mechanical flexible lamellar vertebral body reduction device (Tektona, Spinart).4 We present the case of a 77-year-old woman with back pain after a fall. The radiological investigations show a compressive fracture type AO A1 of T7. After an initial conservative management, the patient presented persistence of the pain with a visual analog scale of 9/10 and progression of the fracture at the 2-week follow-up. On the x-ray, focal kyphosis with vertebra plana was observed. A kyphoplasty for pain management and kyphosis correction was performed. The patient consented to the procedure. A bipedicular approach to the vertebra was performed. The correction of the height was done using 2 mechanical devices. The lamellar device allowed a progressive height restoration of the fracture. On one side, the cannula was used as a support to maintain the correction during the contralateral cementing. The postoperative imaging confirmed a maintained vertebral restoration with kyphosis correction. At 1 year, the patient reported a pain at 1 of 10 on the visual analog scale. In our opinion, this technique is adapted for fresh osteoporotic vertebral compression fractures. Surgical experience in osteoporotic fracture treatment and kyphoplasty is recommended before the management of vertebra plana.

Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations.

Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation-mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis.

Characterization of Commercially Available Human Primary Alveolar Epithelial Cells.

In vitro lung research requires appropriate cell culture models that adequately mimic invivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas-liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC-, KRT8high, and KRT5- cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport invitro.

Prenatal developmental sequences of the esophageal epithelium in the New Zealand white rabbits: Light and electron microscopic analysis.

Several morphogenetic sequences occur during esophageal development and birth defects occur due to defects in foregut morphogenesis. This work aimed to record the cellular events in the morphogenesis of rabbits' esophageal epithelium. On the 16th day of gestation, the esophageal epithelium varied from stratified ciliated columnar to stratified squamous type. The surface epithelium presented mucous cells with mucigen granules of various sizes occupying their supranuclear cytoplasm. Cytoplasmic vacuolation was evident in all layers of the esophageal epithelium at this age. On the 18th gestational day, some light cells could be detected in the middle portion of the epithelium, while others occupied the whole epithelial length. On the 21st day, mucous cells are more frequently observed at the apical esophageal part as well as at the surface epithelium. Numerous elongated dark cells could be distinguished embedded between the basal cells. On the 24th gestational day the number of the mucous cells reached its peak. Reaching the 30th gestational day, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased. Collectively, detection of surface mucous and dark cells together with the non-cornified surface in some regions of the rabbit esophageal epithelium at the end of gestation ensure a postnatal development to reach the adult epithelium essential to sustain the passage of the harsh raw food. Future immunohistochemical studies are recommended to investigate the components of secretions in mucous cells and functional studies to highlight the dark cells significance. RESEARCH HIGHLIGHTS: Esophageal epithelium of fetal rabbit was analyzed by light and transmission microscopy. Surface epithelium presented mucous cells with mucigen granules of various sizes. They reached their maximum number on 24th day then decreased. On the 16th day, cytoplasmic vacuolation was evident in all epithelial layers. On the 21st day, numerous elongated dark cells could be distinguished embedded between the basal cells. Before birth, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased.

Life cell imaging of amiodarone sequestration into lamellar bodies of alveolar type II cells

Amiodarone is widely used to treat cardiac arrhythmias and is very effective in preventing these disorders. However, its use is limited by a wide range of adverse effects, mainly affecting the lungs, and ranging from mild shortness of breath to pulmonary fibrosis. Amiodarone has been shown to accumulate strongly in lung tissue, exceeding its plasma concentration by a hundredfold. However, the site of accumulation and the mechanisms of transport are not fully understood. In this study, we used live cell imaging of primary rat alveolar type II cells to show that amiodarone specifically accumulates in large amounts in lamellar bodies, the surfactant storage organelles. Fluorescence imaging and correlation, and colocalization studies combined with confocal Raman microscopy identified these organelles as a major target for sequestration. Accumulation was rapid, on the order of a few hours, while storage was much more persistent. Partial uptake was observed in chemically fixed, dead cells, or cells treated with bafilomycin A1. Not only was uptake pH dependent, but intraluminal pH, measured with lysosomotropic pH sensitive dyes, was also affected. From these observations and from the physicochemical properties of amiodarone, we propose that passive diffusion, ion-trapping and lipophilic interactions are the main mechanisms for intracellular bioaccumulation. Furthermore, we demonstrate that measurement of amiodarone autofluorescence is highly useful for tracking cellular uptake and sequestration.