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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. [ABSTRACT FROM AUTHOR]

Abstract Background The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. Method GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. Results Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. Conclusions In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

Background: The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. Method: GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. Results: Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. Conclusions: In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs. [ABSTRACT FROM AUTHOR]

The widespread use of disinfectants and inhalation exposure to aerosolized forms is closely associated with adverse health effects on the respiratory system and pulmonary fibrosis, but the mechanism remains unclear. Here, we investigated the time-course pulmonary fibrosis effects of polyhexamethylene guanidine (PHMG) disinfectant inhalation exposure and elucidated its underlying mechanism. Specifically, scRNA-seq analysis revealed an initial increase in epithelial cell numbers after 4 weeks of PHMG exposure during induced pulmonary fibrosis, followed by a subsequent decrease after 8 weeks of exposure. Mechanistically, PHMG disrupted autophagic flux leading to intracellular accumulation and blocked pulmonary surfactant biogenesis in alveolar type II epithelial (AT2) cells both in vitro and in vivo. Furthermore, intervention studies using metformin confirmed that autophagy dysfunction mediated the blockage of pulmonary surfactant biogenesis in AT2 cells, playing a pivotal role in PHMG-induced pulmonary fibrosis. Our elucidation of these toxicological mechanisms provides valuable insights into the pathogenesis of pulmonary fibrosis triggered by environmental PHMG exposure, thereby offering a promising therapeutic target for mitigating and treating PHMG-associated pulmonary fibrosis. [Display omitted] • PHMG disinfectant aerosol induced lung fibrosis characterized by AT2 cell injury. • PHMG aerosol blocked the biogenesis of pulmonary surfactant in AT2 cells. • Autophagy dysfunction mediated the blockage of pulmonary surfactant biogenesis. • PHMG induced autophagy dysfunction in AT2 cells and promoted lung fibrosis. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential. [ABSTRACT FROM AUTHOR]

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.

Borage oil [BO: 40.9% linoleic acid (LNA) and 24.0% γ‐linolenic acid (GLA)] reverses disrupted epidermal lipid barrier in essential fatty acid deficiency (EFAD). We determined the effects of BO on lamellar body (LB) content and LNA and GLA incorporation into epidermal ceramide 1 (CER1) and epidermal ceramide 2 (CER2), major barrier lipids. EFAD was induced in guinea pigs by a diet of 6% hydrogenated coconut oil (HCO) for 10 weeks (group HCO) or 8 weeks followed by 6% BO for 2 weeks (group HCO + BO). LB content and LNA and GLA incorporation into CER1 were higher in group HCO + BO than in group HCO. Small but significant levels of LNA, GLA, and their C20‐metabolized fatty acids [dihomo‐γ‐linolenic acid (DGLA) and arachidonic acid (ARA)] were incorporated into CER2, where ARA was detected at a level lower than LNA, but DGLA incorporation exceeded that for GLA in group HCO + BO. Dietary BO enhanced LB content and differential incorporation of GLA into CER1 and DGLA into CER2. [ABSTRACT FROM AUTHOR]

Lung surfactant accumulates in the lamellar body (LB) via not only the secretory (anterograde) pathway but also the endocytic (retrograde) pathway. Our previous studies suggested that the major surfactant components, phosphatidylcholine and surfactant protein A take independent trafficking routes in alveolar type II cells. Thus, trafficking of surfactant protein B (SP-B), a major hydrophobic surfactant apoprotein, should be re-evaluated by a straightforward method. Radiolabeling of cells and subsequent cell fractionation were employed to pursue the sequential trafficking of newly synthesized SP-B in rabbit alveolar type II cells. The LB fraction was prepared by gradient ultracentrifugation. Immunoprecipitation from the culture medium, total cells, and LB fraction was carried out with anti-SP-B antibody. Newly synthesized [35S]-pro-SP-B (~ 42 kDa) was detected in the cells after 1 h. An ~ 8-kDa mature form of [35S]-SP-B was detected in the cells after 3 h and in the LB after 6 h. Mature [35S]-SP-B was predominant in the cells after 24 h, and the dominant portion was present in the LB. In contrast, only a small amount of mature [35S]-SP-B was present in the culture medium. Molecular processing of ~ 42 kDa [35S]-pro-SP-B and transport to the LB was inhibited by brefeldin A, which disassembles the Golgi apparatus. These results suggest that newly synthesized SP-B is sorted to the LB via the Golgi and stored until exocytosis. This pathway is distinct from the pathways reported for phosphatidylcholine and surfactant protein A. [ABSTRACT FROM AUTHOR]

Background: Lamellar bodies are produced by pneumocyte type II cells in the lung alveoli. Lamellar bodies are present in amniotic fluid in increasing quantities as gestation advances, 1 – 5 µm in size, similar in size to small platelets and can be counted on most electronic cell counters in hematology analyzer. Lamellar body count is useful for prediction of fetal lung maturity and neonatal respiratory distress syndrome. The current gold standard for determination of fetal lung maturity is the evaluation of phospholipids in amniotic fluid samples by thin-layer chromatography, but it is time-consuming and not continuously available at most institutions. In this study we compare Cell Dyn Emerald and Cell Dyn Ruby method, which is expected to be a review for lamellar body count method. Purpose: The aim of this study was to analyze lamellar body count with Cell Dyn Emerald and Cell Dyn Ruby method on preterm birth. Methods : This was a cross sectional study. Thirty three samples study were inpatient’s amniotic fluid with premature rupture of membranes in Obstetry and Gynecology ward emergency room Dr. Soetomo Hospital Surabaya. Lamellar body count was counted with Cell Dyn emerald and Cell Dyn Ruby method. The statistical differences were assessed using the ANOVA test . Results : The results showed significant differences (t=49,04), lamellar body count with Cell Dyn Ruby method was much lower than Cell Dyn Emerald method. The lowest result with Cell Dyn Ruby method was 3.38 x 103/µL and 17 x 103/ µL with Cell Dyn Emerald method. The highest results with Cell Dyn Ruby method was 98,2 x 103/ µL and 221 x 103/ µL with Cell Dyn Emerald method. Conclusion : Lamellar body count with impedance method (Cell Dyn Emerald) is significantly higher than optic method (Cell Dyn Ruby).

Multiwalled carbon nanotubes (MWCNTs) have been widely used in many disciplines and raised great concerns about their negative health impacts, especially environmental and occupational exposure. MWCNTs have been reported to induce fibrotic responses; however, the underlying mechanisms remain largely veiled. Here, we reported that MWCNTs inhalation induced lung fibrosis together with decreased lung compliance, increased elastance in the mice model, and elevated surface tension in vitro . Specifically, MWCNTs increased surface tension by impairing the function of the pulmonary surfactant. Mechanistically, MWCNTs induced lamellar body (LB) dysfunction through autophagy dysfunction, which then leads to surface tension elevated by pulmonary surfactant dysfunction in the context of lung fibrosis. This is a study to investigate the molecular mechanism of MWCNTs-induced lung fibrosis and focus on surface tension. A direct mechanistic link among impaired LBs, surface tension, and fibrosis has been established. This finding elucidates the detailed molecular mechanisms of lung fibrosis induced by MWCNTs. It also highlights that pulmonary surfactants are expected to be potential therapeutic targets for the prevention and treatment of lung fibrosis induced by MWCNTs.

Objective: Plasmin, a relatively unspecific trypsin‐like serine protease, is involved in many physiological and pathological conditions, particularly in dermatoses with barrier impairment. It is secreted as the inactive zymogen plasminogen and is activated to plasmin by plasminogen activators, such as urokinase. There still exists a paucity of data on the precise localization of epidermal plasmin(ogen) within the epidermis and the stratum corneum. The aim of the present study was to get information about its origin and ultrastructural localization within normal human epidermis. Method: We performed immunoelectron transmission electron microscopy immunogold labelling in normal abdominal human skin. Result: Plasmin was only observed in the terminally differentiated cell layers of the epidermis and was largely associated with the corneocyte envelopes and to some extent with the intercellular lipid matrix in the stratum corneum. Conclusion: Our results indicate that in normal human skin, plasmin(ogen) is synthesized by differentiated epidermal keratinocytes of the stratum granulosum and is not serum‐born. [ABSTRACT FROM AUTHOR]

Lysosome‐related organelles (LROs) comprise a diverse group of cell type‐specific, membrane‐bound subcellular organelles that derive at least in part from the endolysosomal system but that have unique contents, morphologies and functions to support specific physiological roles. They include: melanosomes that provide pigment to our eyes and skin; alpha and dense granules in platelets, and lytic granules in cytotoxic T cells and natural killer cells, which release effectors to regulate hemostasis and immunity; and distinct classes of lamellar bodies in lung epithelial cells and keratinocytes that support lung plasticity and skin lubrication. The formation, maturation and/or secretion of subsets of LROs are dysfunctional or entirely absent in a number of hereditary syndromic disorders, including in particular the Hermansky‐Pudlak syndromes. This review provides a comprehensive overview of LROs in humans and model organisms and presents our current understanding of how the products of genes that are defective in heritable diseases impact their formation, motility and ultimate secretion. [ABSTRACT FROM AUTHOR]

We previously showed that topical application of hexoses such as fructose accelerates barrier recovery after disruption. We also showed that various hexoses and polyols interact with phospholipid and alter the phase transition temperature. Thus, we hypothesized that the improvement of barrier recovery by hexoses and polyols might be related to the interaction with phospholipid. Here, we tested this idea by examining the effects of xylitol (a component of some skin-care products) and fructose on lipid dynamics in an epidermal-equivalent model at the single-cell level by means of two-photon microscopy after staining with Laurdan, a fluorescent dye sensitive to the physical properties of its membrane environment. First, we confirmed that topical application of xylitol aqueous solution on tape-stripped human skin accelerated barrier recovery. Then, we examined changes of lipid fluidity in the epidermal-equivalent model after application of water or an aqueous solution of xylitol or fructose. Application of xylitol and/or fructose increased the lipid fluidity in the uppermost part of the stratum granulosum layer, compared to treatment with water alone, and accelerated the exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Our results support the idea that the improvement of epidermal barrier homeostasis upon topical application of xylitol or fructose is due to increased lipid fluidity in the uppermost layer of the stratum granulosum, which enables accelerated release of lipid from the stratum granulosum, thereby improving the lamellar structure and accelerating epidermal permeability barrier recovery. [ABSTRACT FROM AUTHOR]

Background The morbidity and mortality of hyaline membrane dis- ease (HMD) are quite high due to delayed diagnosis and intervention. Commonly, HMD occurs in preterm infants with surfactant deficiency because of lung immaturity. Lung maturity test could be performed using biochemical, biophysical, and amniotic fluid turbidity test. Objective To find out HMD prevalence and the value of shake test and lamellar body concentration in diagnosing HMD in preterm infants. Methods This was a cross-sectional study carried out at Hasan Sadikin Hospital Bandung on preterm infants born during October- December 2001. The shake test was performed using gastric fluid and amniotic fluid while the lamellar body concentration was per- formed using amniotic fluid. Results During the 3-month period, 571 infants were born, of 64 (11.2%) preterm infants, only 41 (64%) fulfilled the inclusion crite- ria; among those preterm infants, 14 (34%) suffered from respira- tory distress and 7 suffered from HMD (prevalence 17%). All HMD cases occurred in infants less than 32 weeks for gestational age. In 7 preterm infants with HMD, the shake test of gastric fluid ob- tained by lavage showed negative results in 3 and +1 in 4 infants; while the shake test of amniotic fluid revealed negative result in 5 and +1 in 2 infants. Lamellar body concentration of amniotic fluid was ≤18,000/ml in all HMD infants. Among three infants less than 32 weeks for gestational age who did not suffer from HMD, +1 shake test of gastric fluid was found in 2 infants and +2 in 1 infant; while shake test of amniotic fluid showed negative result in 1 infant and +1 in 2; the lamellar body concentration of amniotic fluid was ≤18,000/ml in 2 infants and >18,000/ml in 1 infant. Conclusions We concluded that HMD occurred in 17% of preterm infants. The shake test of gastric and amniotic fluids revealed nega- tive or +1 results whereas lamellar body concentration had value of less than or equal to 18,000/mL. More extensive studies are warranted to assess the validity (sensitivity, specificity and predic- tive values) of these measurements

Background To elucidate the impact of fertility treatment on neonatal respiratory outcomes and amniotic lamellar body counts (LBCs) in twin pregnancies. Methods One hundred ninety twin pairs, including 99 dichorionic twin (DCT) and 91 monochorionic twin (MCT) pairs were registered at our institutions. All amniotic fluid samples were obtained from each sac at cesarean section. Samples were analyzed immediately after arrival at the laboratory without centrifugation. We divided the patients into 3 groups: the no therapy group (natural conception), the induced ovulation group (with or without intrauterine insemination), and the assisted reproductive technology (ART) group ( in vitro fertilization or intracytoplasmic sperm injection). Results No statistically significant associations between the fertility treatment and the rates of neonatal RDS/TTN were observed in the whole study population (odds ratio [OR], 0.95; 95% confidence interval [CI], 0.45–2.00), DCT (OR, 0.86; 95%CI, 0.30–2.47), and MCT (OR, 1.45; 95%CI, 0.41–5.11). In addition, there was no association between the fertility treatment and neonatal RDS/TTN in the propensity score analysis of the whole study population (OR, 1.25; 95%CI, 0.57–2.74). Conclusions None of the individual types of fertility treatment had a direct impact on respiratory disorders such as RDS and TTN in twin infants. [ABSTRACT FROM AUTHOR]

Background Whether or not the period of fetal lung maturity differs between twin and singleton pregnancies has not been clarified. We examined whether or not fetal lung maturity and fetal lung absorption are achieved earlier in twin fetuses than in singleton fetuses. Methods We registered 454 singleton pregnancies and 398 twin pregnancies with no congenital abnormalities affecting the respiratory function or neonatal deaths. All patients were delivered by Caesarean section without labor between 24 and 38 gestational weeks. The amniotic fluid samples were analyzed immediately without centrifugation. A multiple logistic regression analysis was performed to explore the relationship between twin pregnancy and neonatal respiratory distress syndrome and transient tachypnea of the newborn (RDS/TTN). Results The rate of RDS/TTN in infants was significantly higher and the lamellar body counts (LBCs) significantly lower in singleton pregnancies than that in twin pregnancies (P < .001). According to a multivariate logistic regression analysis, twin pregnancy (odds ratio, 0.34; 95% confidence interval, 0.22–0.55) was a significant preventive factor for neonatal RDS/TTN. Conclusions We showed that twin fetuses experience more rapid lung maturation and lung fluid absorption than singleton fetuses, as confirmed by the higher LBC values in twin fetuses. [ABSTRACT FROM AUTHOR]

Background Atopic dermatitis is a common skin disease characterized by a Th2 cell-dominant inflammatory infiltrate, elevated serum IgE levels and impaired epidermal barrier function. It is associated to abnormal epidermal lamellar body secretion, producing alteration in lipid composition and extracellular lamellar membrane organization. Objectives The oxazolone-induced atopic dermatitis in hairless mice was used to evaluate in vivo the effect of the application of a lipid system that mimics the morphology, structure and composition of epidermal lamellar bodies. Methods The skin barrier function was evaluated measuring TEWL and skin hydration in vivo . Inflammation was assessed by analysis of serum IgE levels and histological analysis. The microstructure of the intercellular lipid region was also evaluated before and after treatment. Results The skin condition was improved after 10 days of treatment indicated by decreased TEWL, decreased serum IgE levels, reduced epidermal thickness and reduced lymphocyte-dominated infiltrate. However, the treatment did no improve skin hydration. Conclusions The treatment with this lipid system seems to improve the skin condition by reinforcing the barrier function and reducing the skin inflammation. Therefore, the present study provides evidence that this lipid system combining appropriate lipid composition and morphology could be of interest for the development of future treatments for atopic dermatitis. [ABSTRACT FROM AUTHOR]

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general. [ABSTRACT FROM AUTHOR]