General toxicity and screening of reproductive and developmental toxicity following bioaccumulation of oral-dosed perfluorooctanoic acid: Loss of the Golgi apparatus.

Despite its widespread use as a stabilizer across various industries over the past several decades, the health effects of chronic exposure to PFOA are still unclear. We administered PFOA by oral gavage (0, 12.5, 50, and 200 µg/day/mouse, eight groups) to male and female mice for six months. Body weight gain decreased with dose accompanied by increased liver weight, and PFOA altered liver damage-related-blood biochemical indicators and induced pathological lesions, including hepatocellular hypertrophy, cholangiofibrosis, and centrilobular hepatocellular vacuolation. Loss of the Golgi apparatus, formation of lamellar body-like structures, and lipid accumulation were observed in the liver of PFOA-treated mice. We also cohabited five pairs of male and female mice for the last ten days of administration, dosed PFOA to dam up to 28 days after birth, and investigated effects on reproduction and development. The survival rate of pups and the sex ratio of surviving mice decreased significantly at the highest dose. PFOA tissue concentration increased with the dose in the parent mice's liver and the pups' blood and brain. Taken together, we suggest that PFOA primarily affects the liver and reproduction system and that disturbance in lipid metabolism and Golgi's structural stability may be involved in PFOA-induced toxicity.

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

Lamellar body (LB) is a tissue-specific lysosome-related organelle in type II alveolar cells, which is 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 its 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 the reduction of the steady-state level of V-ATPase and the increase of luminal pH of LB. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and bronchoalveolar lavage fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of V-ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.

Pulmonary Surfactant Homeostasis Dysfunction Mediates Multiwalled Carbon Nanotubes Induced Lung Fibrosis via Elevating Surface Tension.

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.

GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells.

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.

An inducible tricolor reporter mouse for simultaneous imaging of lysosomes, mitochondria and microtubules.

Cell-type-specific use of the same DNA blueprint generates diverse cell types. Such diversity must also be executed via differential deployment of the same subcellular machinery. However, our understanding of the size, distribution, and dynamics of subcellular machinery in native tissues, and their connection to cellular diversity, remain limited. We generate and characterize an inducible tricolor reporter mouse, dubbed "kaleidoscope", for simultaneous imaging of lysosomes, mitochondria and microtubules in any cell type and at a single cell resolution. The expected subcellular compartments are labeled in culture and in tissues with no impact on cellular and organismal viability. Quantitative and live imaging of the tricolor reporter captures cell-type-specific organelle features and kinetics in the lung, as well as their changes after Sendai virus infection. Yap/Taz mutant lung epithelial cells undergo accelerated lamellar body maturation, a subcellular manifestation of their molecular defects. A comprehensive toolbox of reporters for all subcellular structures is expected to transform our understanding of cell biology in tissues.

An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients.

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.

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites.

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as "giant lamellar bodies" (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.

Lamellar body count: Marker for foetal lung maturation promoted by intra-amniotic infection and/or inflammation.

OBJECTIVE: There is evidence indicating that the risk of respiratory distress syndrome is reduced in preterm neonates exposed to intra-amniotic infection and/or inflammation. We hypothesised that foetal lung maturation promoted by intra-amniotic infection and/or inflammation results in elevated lamellar body count (LBC) in amniotic fluid (AF). This study aimed to determine the relationship between LBC in AF and intra-amniotic infection and/or inflammation in patients with threatened preterm birth. STUDY DESIGN: This was a retrospective cohort study of patients with threatened preterm birth. A total of 104 consecutive pregnant women underwent amniocentesis in the early preterm period [gestational age < 34 weeks] to evaluate intra-amniotic infection and/or inflammation and foetal lung maturity. Intra-amniotic infection was confirmed by positive AF culture results for aerobic/anaerobic bacteria, fungi, and genital mycoplasma. Intra-amniotic inflammation was defined as a positive AF matrix metalloproteinase-8 rapid test. Outcomes of the study population were compared according to LBC in AF using a cut-off of 15,000/mm3. RESULTS: The rates of elevated LBC and intra-amniotic infection and/or inflammation were 23% (24/104) and 52% (54/104), respectively. The median LBC was significantly higher in patients with intra-amniotic infection and/or inflammation than in those without [median LBC, 9,000/mm3 (interquartile range, IQR: 3,000-39,000) vs. 3,000/mm3 (IQR: 2,750-5,000), p < 0.001]. Intra-amniotic infection and/or inflammation was observed in 96% (23/24) of patients with elevated LBC and 39% (31/80) of patients without elevated LBC (p < 0.001). On multivariable analysis, the presence of intra-amniotic infection and/or inflammation was significantly associated with elevated LBC with an odds ratio (OR) of 66.0 [95% confidence interval (CI) 6.6-664.4, p < 0.001], even after accounting for gestational age at amniocentesis being a significantly related factor for predicting elevated LBC with an OR of 1.5 (95% CI 1.1-2.0, p = 0.004). CONCLUSION: LBC elevation was independently associated with the presence of intra-amniotic infection and/or inflammation in women with early threatened preterm birth (gestational age < 34 weeks). This finding may support the view that an intra-amniotic inflammatory response promotes foetal lung maturation that can be detected by elevated LBC in AF.

Pulmonary inflammation and cellular responses following exposure to benzalkonium chloride: Potential impact of disrupted pulmonary surfactant homeostasis.

Benzalkonium chloride (BKC) is a prototypical quaternary ammonium disinfectant. Previously, we suggested a no lethal dose level (0.005%) and an LD50 range (0.5-0.05%) of BKC following a single pharyngeal aspiration. Herein, we exposed BKC repeatedly by pharyngeal aspiration for 14 days (0.005 and 0.01%, female mice, total five times with interval of two days, 5 mice/group) and 28 days (0, 0.001, 0.005, and 0.01%, male and female mice, weekly, 16 mice/sex/group). Death following 14 days-repeated exposure did not occur. Meanwhile, chronic pathological lesions were observed in the lung tissues of mice exposed to BKC for 28 days. The total number of bronchial alveolar lavage cells increased, and pulmonary homeostasis of immunologic messenger molecules was disturbed. Following, we investigated BKC-induced cellular responses using human bronchial epithelial cells. The cytotoxicity increased rapidly with concentration. Lysosomal volume, NO production, and lipid peroxidation increased in BKC-treated cells, whereas intracellular ROS level decreased accompanying structural and functional damage of mitochondria. We also found that BKC affected the expression level of immune response, DNA damage, and amino acid biosynthesis-related molecules. More interestingly, lamellar body- and autophagosome-like structures were notably observed in cells exposed to BKC, and necrotic and apoptotic cell death were identified accompanying cell accumulation in the G2/M phase. Therefore, we suggest that repeated respiratory exposure of BKC causes pulmonary inflammation and lung tissue damage and that dead and damaged cells may contribute to the inflammatory response. In addition, the formation process of lamellar body-like structures may function as a key toxicity mechanism.

The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells.

The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 (Atg7) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood.

Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis.

Alterations of the lipid profile of the stratum corneum have an important role in the pathogenesis of atopic dermatitis (AD) because they contribute to epidermal barrier impairment. However, they have not previously been envisioned as a cellular response to altered metabolic requirements in AD epidermis. In this study, we report that the lipid composition in the epidermis of flaky tail, that is, ft/ft mice mimics that of human lesional AD (ADL) epidermis, both showing a shift toward shorter lipid species. The amounts of C24 and C26 free fatty acids and C24 and C26 ceramides-oxidized exclusively in peroxisomes-were reduced in the epidermis of ft/ft mice despite increased lipid synthesis, similar to that seen in human ADL edpidermis. Increased ACOX1 protein and activity in granular keratinocytes of ft/ft epidermis, altered lipid profile in human epidermal equivalents overexpressing ACOX1, and increased ACOX1 immunostaining in skin biopsies from patients with ADL suggest that peroxisomal ß-oxidation significantly contributes to lipid signature in ADL epidermis. Moreover, we show that increased anaerobic glycolysis in ft/ft mouse epidermis is essential for keratinocyte proliferation and adenosine triphosphate synthesis but does not contribute to local inflammation. Thus, this work evidenced a metabolic shift toward enhanced peroxisomal ß-oxidation and anaerobic glycolysis in ADL epidermis.

Modeling of lung phenotype of Hermansky-Pudlak syndrome type I using patient-specific iPSCs.

BACKGROUND: Somatic cells differentiated from patient-specific human induced pluripotent stem cells (iPSCs) could be a useful tool in human cell-based disease research. Hermansky-Pudlak syndrome (HPS) is an autosomal recessive genetic disorder characterized by oculocutaneous albinism and a platelet dysfunction. HPS patients often suffer from lethal HPS associated interstitial pneumonia (HPSIP). Lung transplantation has been the only treatment for HPSIP. Lysosome-related organelles are impaired in HPS, thereby disrupting alveolar type 2 (AT2) cells with lamellar bodies. HPSIP lungs are characterized by enlarged lamellar bodies. Despite species differences between human and mouse in HPSIP, most studies have been conducted in mice since culturing human AT2 cells is difficult. METHODS: We generated patient-specific iPSCs from patient-derived fibroblasts with the most common bi-allelic variant, c.1472_1487dup16, in HPS1 for modeling severe phenotypes of HPSIP. We then corrected the variant of patient-specific iPSCs using CRISPR-based microhomology-mediated end joining to obtain isogenic controls. The iPSCs were then differentiated into lung epithelial cells using two different lung organoid models, lung bud organoids (LBOs) and alveolar organoids (AOs), and explored the phenotypes contributing to the pathogenesis of HPSIP using transcriptomic and proteomic analyses. RESULTS: The LBOs derived from patient-specific iPSCs successfully recapitulated the abnormalities in morphology and size. Proteomic analysis of AOs involving iPSC-derived AT2 cells and primary lung fibroblasts revealed mitochondrial dysfunction in HPS1 patient-specific alveolar epithelial cells. Further, giant lamellar bodies were recapitulated in patient-specific AT2 cells. CONCLUSIONS: The HPS1 patient-specific iPSCs and their gene-corrected counterparts generated in this study could be a new research tool for understanding the pathogenesis of HPSIP caused by HPS1 deficiency in humans.

Epidermal Lamellar Body Biogenesis: Insight Into the Roles of Golgi and Lysosomes.

Epidermal lamellar bodies (eLBs) are secretory organelles that carry a wide variety of secretory cargo required for skin homeostasis. eLBs belong to the class of lysosome-related organelles (LROs), which are cell-type-specific organelles that perform diverse functions. The formation of eLBs is thought to be related to that of other LROs, which are formed either through the gradual maturation of Golgi/endosomal precursors or by the conversion of conventional lysosomes. Current evidence suggests that eLB biogenesis presumably initiate from trans-Golgi network and receive cargo from endosomes, and also acquire lysosome characteristics during maturation. These multistep biogenesis processes are frequently disrupted in human skin disorders. However, many gaps remain in our understanding of eLB biogenesis and their relationship to skin diseases. Here, we describe our current understanding on eLB biogenesis with a focus on cargo transport to this LRO and highlight key areas where future research is needed.

No air without autophagy: autophagy is important for lung and swim bladder inflation.

Macroautophagy is a catabolic process critical for the degradation of intracellular material, but its physiological functions in vertebrates are not fully understood. Here, we discuss our recent finding that macroautophagy plays a role in lamellar body maturation. The lamellar body is a lysosome-related organelle and stores phospholipid-containing surfactant complexes that reduce the surface tension of the air-water interface in order to inflate the airspace in lungs and swim bladders. In the epithelial cells of these organs, autophagosomes fuse with immature lamellar bodies to increase their size and lipid contents. This function is essential for respiration after birth in mice and for maintaining buoyancy in zebrafish. These findings unveil a novel function of macroautophagy in the maturation of surfactant-containing lamellar bodies.

Borage Oil Enhances Lamellar Body Content and Alters Fatty Acid Composition of Epidermal Ceramides in Essential Fatty Acid-Deficient Guinea Pigs.

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.

Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder.

Autophagy is an intracellular degradation system, but its physiological functions in vertebrates are not yet fully understood. Here, we show that autophagy is required for inflation of air-filled organs: zebrafish swim bladder and mouse lung. In wild-type zebrafish swim bladder and mouse lung type II pulmonary epithelial cells, autophagosomes are formed and frequently fuse with lamellar bodies. The lamellar body is a lysosome-related organelle that stores a phospholipid-containing surfactant complex that lines the air-liquid interface and reduces surface tension. We find that autophagy is critical for maturation of the lamellar body. Accordingly, atg-deficient zebrafish fail to maintain their position in the water, and type-II-pneumocyte-specific Fip200-deficient mice show neonatal lethality with respiratory failure. Autophagy suppression does not affect synthesis of the surfactant phospholipid, suggesting that autophagy supplies lipids and membranes to lamellar bodies. These results demonstrate an evolutionarily conserved role of autophagy in lamellar body maturation.

Sodium Glucose Co-Transporter 2 Inhibitor Ameliorates Autophagic Flux Impairment on Renal Proximal Tubular Cells in Obesity Mice.

Obesity is supposed to cause renal injury via autophagy deficiency. Recently, sodium glucose co-transporter 2 inhibitors (SGLT2i) were reported to protect renal injury. However, the mechanisms of SGLT2i for renal protection are unclear. Here, we investigated the effect of SGLT2i for autophagy in renal proximal tubular cells (PTCs) on obesity mice. We fed C57BL/6J mice with a normal diet (ND) or high-fat and -sugar diet (HFSD) for nine weeks, then administered SGLT2i, empagliflozin, or control compound for one week. Each group contained N = 5. The urinary N-acetyl-beta-d-glucosaminidase level in the HFSD group significantly increased compared to ND group. The tubular damage was suppressed in the SGLT2i-HFSD group. In electron microscopic analysis, multi lamellar bodies that increased in autophagy deficiency were increased in PTCs in the HFSD group but significantly suppressed in the SGLT2i group. The autophagosomes of damaged mitochondria in PTCs in the HFSD group frequently appeared in the SGLT2i group. p62 accumulations in PTCs were significantly increased in HFSD group but significantly suppressed by SGLT2i. In addition, the mammalian target of rapamycin was activated in the HFSD group but significantly suppressed in SGLT2i group. These data suggest that SGLT2i has renal protective effects against obesity via improving autophagy flux impairment in PTCs on a HFSD.

Trafficking of newly synthesized surfactant protein B to the lamellar body in alveolar type II cells.

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.

Diagnostic accuracy of lamellar body count as a predictor of fetal lung maturity: A systematic review and meta-analysis.

OBJECTIVE: This study aimed to synthesize evidence from published studies about the diagnostic accuracy of lamellar body count (LBC) as a predictor of fetal lung maturity. STUDY DESIGN: We searched Medline (via PubMed), EBSCO, Web of Science, Scopus and the Cochrane Library for relevant published studies assessing the accuracy of LBC as a predictor of fetal lung maturity. Studies were classified according to the counting essays, centrifugation protocols, and the reported optimum cut off values. Data of the true positive, true negative, false positive, and false negative were extracted and analyzed to calculate the overall sensitivity and specificity of the LBC. RESULTS: Thirty-one studies were included in the final analysis. Fourteen studies reported data for centrifuged amniotic fluid (AF) samples, 13 studies reported data for uncentrifuged samples, and four studies did not have enough information about whether centrifugation was done. LBC showed an area under the curve >80% in diagnosing lung immaturity with variable cut off values. Pooled analysis showed that LBC a 100% specificity to exclude respiratory distress syndrome (RDS) at a cut off value of 15,000 and 100% sensitivity to diagnose RDS at a cut off value of 55,000. CONCLUSION: Cases with LBC < 15,000 are considered to have lung immaturity while cases with LBC > 45,000 in centrifuged AF samples or >55,000 in uncentrifuged AF samples are likely to have mature lungs. Cases with LBC ranging between these maturity and immaturity limits should be considered for further evaluation by other lung maturity tests.

Expression and ultrastructural localization of plasmin(ogen) in the terminally differentiated layers of normal human epidermis.

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.

OBJECTIF: Plasmine, une relativement peu spécifique ' trypsin-like' protéase sérine, participe aux plusieurs processus physiologiques et pathologiques et, plus particulièrement, à la physiopathologie des dermatoses caractérisées par l'altération de la barrière de perméabilité. Elle est sécrétée sous forme d'un zymogene inactif, plasminogène, et devient activée par les activateurs du plasminogène, telle urokinase. A l'heure actuelle, on manque de précision quant à la localisation de plasmine (ou son précurseur) dans l'épiderme et le stratum corneum. Le but du présent travail a été de d'apporter l'information sur la provenance et la localisation ultrastructurale de plasmine/plasminogène présents dans l'épiderme humain. MÉTHODE: L'étude ultrastructurale de l'épiderme humain normal (plastie abdominale) a fait appel à l'immunomarquage à l'or colloïdal sur coupes ultrafines des tissus inclus à froid dans des résines acryliques. RÉSULTAT: L'anticorps monoclonal anti -plasmine/plasminogène a détecté l'antigène situé exclusivement dans la partie la plus différenciée de l'épiderme et persistant dans la couche cornée. Il n'y a pas eu de réactivité dans les couches épineuse et basale. Le marquage a été prédominant sur les enveloppes cornifiées des kératinocytes granuleux et cornéocytes. Des foyers du marquage ont été également présents dans le cytoplasme et les espaces intercellulaires de la couche granuleuse, ainsi que dans la matrice lipidique de la couche cornée profonde. CONCLUSION: Nos résultats indiquent la production de novo de plasmine/plasminogène dans les kératinocytes le plus différenciés et ne suggèrent pas l'origine sérique de cette enzyme dans l'épiderme.