Alveolar Septae Research Articles

Minute lesions of alveolar damages in lungs of patients with stable chronic obstructive pulmonary disease

Objective: To reveal the mechanism underlying recruitment of neutrophils in chronic obstructive pulmonary disease (COPD) lungs and to investigate the role of minute lesions of alveolar damage (MLADs) in emphysema development. Methods: Seventy-four lobes from 74 patients with stable COPD, and 78 lobes from 78 patients without COPD (controls) were immunohistochemically examined. Results: MLADs presented as microscopic foci of inflammatory lung injury where alveolar epithelial cells had fragmented and disappeared, and ring-like or tubelike capillary structures had disappeared from the alveolar septae. MLADs were detected in 11 of 74 COPD patients. Tumor necrosis factor (TNF) -α+ macrophages and hypoxia inducible factor (HIF)-2α+ macrophages were detected in 100% of patients in the smoking group with or without COPD and in all patients with COPD exhibiting MLADs. The numbers of neutrophils in alveolar septae and alveolar spaces were significantly larger in the COPD smoking group and non-COPD smoking group than in the non-COPD non-smoking group, and the numbers of neutrophils were tend to be larger in and around MLADs than in lung tissues located away from MLADs in smoking patients with COPD. Masson body-like tissues estimated to be organizations of exudates as well as mild interstitial fibrosis estimated to represent the fibroproliferative phase of MLADs were observed in patients with COPD and smoking patients without COPD. Conclusion: These findings suggest the followings: (1). HIF-2αA¯Â¼Â‹macrophages and TNF-αA¯Â¼Â‹macrophages induced by hypoxia caused by smoking play an important role in the recruitment of neutrophils, (2). MLADs develop in lungs in which large number of neutrophils have been recruited probably by deterioration of hypoxia and play an important role in the development of subsequent full-scale emphysema.

Glufosinate aerogenic exposure induces glutamate and IL-1 receptor dependent lung inflammation.

Glufosinate-ammonium (GLA), the active component of an herbicide, is known to cause neurotoxicity. GLA shares structural analogy with glutamate. It is a powerful inhibitor of glutamine synthetase (GS) and may bind to glutamate receptors. Since these potentials targets of GLA are present in lung and immune cells, we asked whether airway exposure to GLA may cause lung inflammation in mice. A single GLA exposure (1mg/kg) induced seizures and inflammatory cell recruitment in the broncho-alveolar space, and increased myeloperoxidase (MPO), inducible NO synthase (iNOS), interstitial inflammation and disruption of alveolar septae within 6-24h. Interleukin 1β (IL-1β) was increased and lung inflammation depended on IL-1 receptor 1 (IL-1R1). We demonstrate that glutamate receptor pathway is central, since the N-methyl-D-aspartate (NMDA) receptor inhibitor MK-801 prevented GLA-induced lung inflammation. Chronic exposure (0.2mg/kg 3× per week for 4weeks) caused moderate lung inflammation and enhanced airway hyperreactivity with significant increased airway resistance. In conclusion, GLA aerosol exposure causes glutamate signalling and IL-1R-dependent pulmonary inflammation with airway hyperreactivity in mice.

Expression Of The Surfactant Protein C Trafficking Mutant SP‐CI73T In Vivo Results In Alterations In Alveolar Type 2 Cell Quality Control and Parenchymal Remodeling In Mice

Mutations in the gene encoding for the alveolar type 2 cell (AT2) specific protein, Surfactant Protein C (SFTPC; SP‐C), have been linked to both familial and sporadic variants of diffuse parenchymal lung disease (DPLD) in adults and children. The most common DPLD‐associated SFTPC mutation, 218T>C, results in a missense substitution of isoleucine to threonine at position 73 [I73T] in the SP‐C proprotein [proSP‐C]). Using cell lines stably expressing SP‐C isoforms, we had previously shown that in contrast to wild‐type SP‐C, the mutant SP‐CI73T proprotein is mistrafficked to the plasma membrane, accumulates in endosomal compartments, and produces a late block in macroautophagy. To define the consequences of expression of SP‐CI73T for AT2 cells in vivo, recombineering strategies were employed to selectively knock‐in an HA‐tagged mouse SP‐CI73T sequence into the sftpc locus. The founder line, whose alleles retained the PGK‐neomycin (PGK‐neo) cassette in intron 1 used for ES cell selection, demonstrates hypomorphic expression of mutant sftpc mRNA (~ 10% of wild‐type levels) permitting the animals to be born alive but subjected to the accumulation of mutant HA‐proSP‐CI73T in a time dependent fashion. Morphologically, these animals exhibit normal post‐natal alveolarization; however, by 16 weeks of age, they develop age‐dependent diffuse parenchymal lung remodeling characterized by a cellular and mesenchymal expansion of alveolar septae. By fluorescence IHC, abnormal trafficking of mutant HA‐SP‐CI73T proprotein was manifested by its prominent localization in plasma membrane and sub‐plasma membrane vesicular compartments. Subcellular fractionation and Western blotting confirmed that proSP‐CI73T was misprocessed and excluded from lamellar bodies of AT2 cells. Autophagic flux assays using AT2 cells isolated from 16 and 32 week old SP‐CI73T mice revealed aberrant LC3 (atg8) interconversion accompanied by increases in p62 expression both of which were more sensitive to bafilomycin A1 treatment than wild‐type AT2 cells. Ultrastructurally, transmission EM showing hypertrophic SP‐CI73T AT2 cells with large autophagosomal vacuoles containing mitochondria and proteinacious debris confirmed the late block in macroautophagy. Despite a 2 fold compensatory increase in total 26S proteasomal (chymotrypsin‐like) enzymatic activity and biomass, treatment of SP‐CI73T expressing cells with MG132 did not alter the level of mutant proSP‐CI73T. We conclude that expression of aberrantly trafficked and misprocessed SP‐CI73T in AT2 cells in vivo results in its exclusion from proteasomal degradation and inhibition of macroautophagy. These results offer proof of concept that disruption of epithelial cell quality control could contribute to parenchymal lung remodeling through alterations in AT2 cell homeostasis and function.Support or Funding InformationSupported by NIH HL 119436, VA Merit Award BX001176, and an Albert Rose Career Development Award from the Pulmonary Fibrosis Foundation (all to MFB).

Commentary: A Breath of Fresh Air on the Mesenchyme: Impact of Impaired Mesenchymal Development on the Pathogenesis of Bronchopulmonary Dysplasia.

Commentary: A Breath of Fresh Air on the Mesenchyme: Impact of Impaired Mesenchymal Development on the Pathogenesis of Bronchopulmonary Dysplasia.

A three-dimensional human model of the fibroblast activation that accompanies bronchopulmonary dysplasia identifies Notch-mediated pathophysiology.

Bronchopulmonary dysplasia (BPD) is a leading complication of premature birth and occurs primarily in infants delivered during the saccular stage of lung development. Histopathology shows decreased alveolarization and a pattern of fibroblast proliferation and differentiation to the myofibroblast phenotype. Little is known about the molecular pathways and cellular mechanisms that define BPD pathophysiology and progression. We have developed a novel three-dimensional human model of the fibroblast activation associated with BPD, and using this model we have identified the Notch pathway as a key driver of fibroblast activation and proliferation in response to changes in oxygen. Fetal lung fibroblasts were cultured on sodium alginate beads to generate lung organoids. After exposure to alternating hypoxia and hyperoxia, the organoids developed a phenotypic response characterized by increased α-smooth muscle actin (α-SMA) expression and other genes known to be upregulated in BPD and also demonstrated increased expression of downstream effectors of the Notch pathway. Inhibition of Notch with a γ-secretase inhibitor prevented the development of the pattern of cellular proliferation and α-SMA expression in our model. Analysis of human autopsy tissue from the lungs of infants who expired with BPD demonstrated evidence of Notch activation within fibrotic areas of the alveolar septae, suggesting that Notch may be a key driver of BPD pathophysiology.

Pulmonary Intravascular Lymphomatosis: Clinical, CT, and PET Findings, Correlation of CT and Pathologic Results, and Survival Outcome.

Purpose To describe clinical, computed tomographic (CT), and positron emission tomographic (PET) features, correlation of CT and pathologic results, and survival of patients with pulmonary intravascular lymphomatosis. Materials and Methods The institutional review board approved this retrospective study with waiver of patient consent. Forty-two patients with pulmonary intravascular lymphomatosis were identified, 11 (26%) of whom showed lung involvement. CT features were correlated with histopathologic results. Clinical and survival outcomes were compared between patients with and those without pulmonary involvement by adopting the χ(2), Student t, or Kaplan-Meier analysis with log-rank tests. Results At clinical presentation, all 11 patients showed B symptoms (systemic symptoms of fever, night sweats, and weight loss), 10 had respiratory and four had neurologic symptoms, and two had skin lesions. Patients received cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy with (n = 5) or without (n = 6) rituximab, and seven (64%) patients died. Patients with lung involvement showed reduced overall and recurrence-free survival (median; 10.8 and 18.9 months, respectively) compared with those without lung involvement (median, 18.4 and 31.0 months, respectively) (P = .338 and .065, respectively). The most common CT abnormality was bilateral ground-glass opacity (GGO, n = 10), with increased fluorodeoxyglucose uptake at PET/CT (seven of seven patients). GGO correlated histopathologically with the expanded alveolar septal vasculatures and perivascular spaces filled with neoplastic lymphoid cells. Conclusion Pulmonary intravascular lymphomatosis appeared as bilateral GGO on CT images, with increased fluorodeoxyglucose uptake on PET/CT images. GGO on CT images correlated with the area of expanded alveolar septae because of distended vessels filled with neoplastic lymphoid cells. (©) RSNA, 2016 Online supplemental material is available for this article.

A three-dimensional study of alveologenesis in mouse lung

Alveologenesis is the final step of lung maturation, which subdivides the alveolar region of the lung into smaller units called alveoli. Each of the nascent dividers serves as a new gas-exchange surface, and collectively they drastically increase the surface area for breathing. Disruption of alveologenesis results in simplification of alveoli, as is seen in premature infants diagnosed with bronchopulmonary dysplasia (BPD), a prevalent lung disease that is often associated with lifelong breathing deficiencies. To date, a majority of studies of alveologenesis rely on two-dimensional (2D) analysis of tissue sections. Given that an overarching theme of alveologenesis is thinning and extension of the epithelium and mesenchyme to facilitate gas exchange, often only a small portion of a cell or a cellular structure is represented in a single 2D plane. Here, we use a three-dimensional (3D) approach to examine the structural architecture and cellular composition of myofibroblasts, alveolar type 2 cells, elastin and lipid droplets in normal as well as BPD-like mouse lung. We found that 2D finger-like septal crests, commonly used to depict growing alveolar septae, are often artifacts of sectioning through fully established alveolar walls. Instead, a more accurate representation of growing septae are 3D ridges that are lined by platelet-derived growth factor receptor alpha (PDGFRA) and alpha smooth muscle actin (α-SMA)-expressing myofibroblasts, as well as the elastin fibers that they produce. Accordingly in 3D, both α-SMA and elastin were each found in connected networks underlying the 3D septal ridges rather than as isolated dots at the tip of 2D septal crests. Analysis through representative stages of alveologenesis revealed unappreciated dynamic changes in these patterns. PDGFRA-expressing cells are only α-SMA-positive during the first phase of alveologenesis, but not in the second phase, suggesting that the two phases of septae formation may be driven by distinct mechanisms. Thin elastin fibers are already present in the alveolar region prior to alveologenesis, suggesting that during alveologenesis, there is not only new elastin deposition, but also extensive remodeling to transform thin and uniformly distributed fibers into thick cables that rim the nascent septae. Analysis of several genetic as well as hyperoxia-induced models of BPD revealed that the myofibroblast organization is perturbed in all, regardless of whether the origin of defect is epithelial, mesenchymal, endothelial or environmental. Finally, analysis of relative position of PDGFRA-positive cells and alveolar type 2 cells reveal that during alveologenesis, these two cell types are not always adjacent to one another. This result suggests that the niche and progenitor relationship afforded by their close juxtaposition in the adult lung may be a later acquired property. These insights revealed by 3D reconstruction of the septae set the foundation for future investigations of the mechanisms driving normal alveologenesis, as well as causes of alveolar simplification in BPD.

The effect of prenatal maternal infection on respiratory function in mouse offspring: evidence for enhanced chemosensitivity.

Systemic maternal inflammation is implicated in preterm birth and bronchopulmonary dysplasia (BPD) and may induce morbidities including reduced pulmonary function, sleep-disordered breathing, and cardiovascular disorders. Here we test the hypothesis that antenatal maternal inflammation per se causes altered alveolar development and increased chemoreflex sensitivity that persists beyond infancy. Pregnant C57BL/6 mice were administered lipopolysaccharide (LPS) (150 μg/kg ip) to induce maternal inflammation or saline (SHAM) at embryonic day 16 (randomized). Pups were weighed daily. On days 7, 28, and 60 (D07, D28, and D60), unrestrained wholebody plethysmography quantified ventilation and chemoreflex responses to hypoxia (10%), hypercapnia (7%), and asphyxia (hypoxic hypercapnia). Lungs were harvested to quantify alveolar number, size, and septal thickness. LPS pups had reduced baseline ventilation per unit bodyweight (∼40%, P < 0.001) vs. SHAM. LPS increased ventilatory responses to hypoxia (D07: 66% vs. 28% increase in ventilation; P < 0.001) hypercapnia (170% vs. 88%; P < 0.001), and asphyxia (249% vs. 154%; P < 0.001); hypersensitive hypoxic responsiveness persisted until D60 (P < 0.001). LPS also increased apnea frequency (P < 0.01). LPS caused thicker alveolar septae (D07, P < 0.001), diminished alveolar number (D28, P < 0.001) vs. SHAM, but effects were minimal by D60. Pups delivered from mothers exposed to antenatal inflammation exhibit deficits in lung structure and hypersensitive responses to respiratory stimuli that persist beyond the newborn period. Antenatal inflammation may contribute to impaired gas exchange and unstable breathing in newborn infants and adversely affect long-term health.

Left ventricular obstruction with restrictive inter-atrial communication leads to retardation in fetal lung maturation.

Intact atrial septum or highly restrictive inter-atrial communication (I/HRAS) combined with either severe aortic stenosis (SAS) or hypoplastic left heart syndrome (HLHS), respectively, is associated with adverse outcome. This study focusses on changes in alveolo-septal lung parenchyma due to increased left atrial pressure. In a retrospective cross-sectional autoptic study, we investigated fetal/neonatal lung specimens of 18 patients with SAS/HLHS with I/HRAS, 11 patients with SAS/HLHS and unrestrictive inter-atrial communications and 18 controls. Pulmonary maturation was investigated by means of morphometric and immunohistochemical analyses. In a comparison of all three groups, alveolo-capillary membrane maturation was significantly disturbed in I/HRAS fetuses from week 23 of pregnancy on. I/HRAS lungs showed angiomatoid hyper-capillarisation and significantly wider inter-airspace mesenchyme. Differences in width ranged between 34.58 µm (95% CI: 11.41-57.75 µm) and 46.74 µm (95% CI: 13.97-79.50 µm) in the second and third trimesters. In I/HRAS infants with HLHS, inter-airspace mesenchymal diameters steadily normalised with age; however, significant fibroelastosis of alveolar septae developed. Fetal lung maturation with respect to alveolo-capillary membrane formation is severely disordered in patients with SAS/HLHS with I/HRAS. Our findings indicate that, from a morphological point of view, timing of fetal invention in fetuses with I/HRAS should be fixed within the second trimester of pregnancy.

Structural Remodeling of the Post‐pneumonectomy Lung is Characterized by Septal Retraction and Alveolar Duct Dilation

Murine pneumonectomy (PNX) is an established model of adult compensatory lung growth, involving structural remodeling of the peripheral parenchyma. Following left PNX, positron emission tomography and computerized tomography have demonstrated heterogeneous growth patterns within the remaining right lung. To characterize the structural changes associated with these observations, we analyzed histological sections of the right lung between 3 and 22 days after PNX. Within 3 days of PNX, alveolar septae were significantly shorter (p<0.05) in a majority of alveolar ducts, while septal angle remained unchanged. Septal retraction resulted in an apparent dilation of the alveolar duct with a significantly decreased alveolar duct diameter ratio (Dout:Din; p<0.01). The greatest percentage of dilated alveolar ducts was seen at day 3, with 46% of alveolar ducts in the most dilated alveolar duct ratio quintile. Heterogeneous septal lengthening was observed between 4 and 22 days after PNX, with average septal length retu...

Pathological findings in wild harvested dugongs Dugong dugon of central Torres Strait, Australia.

The dugong Dugong dugon is classified as Vulnerable to extinction but may be endangered in some regions. Cause of death in stranded dugongs has not been determined in a large proportion of animals examined, with investigations hindered by limited information on dugong health and diseases, and paucity of knowledge of common or endemic pathological findings. Here we describe pathological findings in harvested dugongs from the relatively pristine area of central Torres Strait, and we characterise lesions attributable to drowning. Other recorded lesions were mild and predominated by host reaction to the presence of trematodes within the gastrointestinal tracts, liver and pancreas. Ascarid worm burdens were low in comparison to dugongs from developed coastlines. Hepatocellular lipofuscin and ferritin pigmentation were commonly observed, more pronounced in livers of older animals and concurrent with periportal and bridging fibrosis. Lesions attributable to drowning included incomplete collapse of lungs, dorsal or diffuse pulmonary congestion, mild intra-alveolar haemorrhage and oedema, mild interstitial oedema and rupture of peripheral alveolar septae with acute myofibre fragmentation and degeneration. No accumulation of foam or aspiration of water or particulate matter was observed, suggesting that dugongs 'dry drown'. Morphometric features of normal spleen are also presented. Characterisation of common pathological findings and those attributable to drowning in this species will aid in the interpretation of post mortem findings for the significant number of dugongs found deceased along urbanised coastlines.

Pulmonary capillary hemangiomatosis: a rare cause of pulmonary hypertension.

Pulmonary capillary hemangiomatosis is a rare cause of pulmonary hypertension characterized by extensive proliferation of pulmonary capillaries within alveolar septae. Clinical presentation is nonspecific and includes dyspnea, cough, chest pain, and fatigue. Radiology shows diffuse centrilobular ground-glass opacities. Pulmonary capillary hemangiomatosis is clinically and radiographically indistinguishable from peripheral venoocclusive disease, making microscopic diagnosis essential. Histologically, pulmonary capillary hemangiomatosis shows an abnormal proliferation of small, thin-walled capillaries that expand the alveolar septae. The endothelial cells that comprise these lesions are cytologically bland and show no mitotic activity. Pulmonary capillary hemangiomatosis is important to recognize because prostacyclin therapy, a mainstay in the treatment of pulmonary hypertension, has been reported to cause sudden respiratory distress and death in these patients. Prognosis of this disease remains poor, and the only definitive treatment is lung transplantation.

S98 A Novel Human Model To Study Alveolar Injury And Repair

Introduction The development of regenerative therapies holds promise for the future treatment of parenchymal lung diseases. However, encouraging preclinical data from animal models have translated poorly in clinical trials. The cellular and molecular response to lung injury is difficult to study in man. To address this fundamental question, we have developed a novel in vitro human model. Precision cut lung slice (PCLS) culture is a well-established tool in airway biology and pharmacology. Here, we demonstrate lung parenchyma can be maintained and manipulatedin vitro generating a tractable model, which allows study of lung injury and repair in man. Methods PCLS (500 µm) were generated from agarose-inflated lung lobes from human lungs maintained ex-vivo by perfusion and ventilation (EVLP). The slices were cultured in serum-free medium in a rotating incubator (37oC, 5% CO2) and analysed at days 1, 3 and 7. Cell specific immunofluorescence markers were used to identify smooth muscle, type I and type II alveolar epithelial cells (AT1, AT2), vascular endothelial cells and proliferating cells (using αSMA, Aquaporin5, ProSPC, PECAM1 and Phospho-histone H3 respectively). Slice viability was confirmed using MitoTracker, LDH and Live/Dead assays. Results All of the expected cell types were identified in PCLS by immunofluorescence demonstrating that human PCLS maintained cellular differentiation in culture. Pro-SPC was predominant in the alveolar wall cells, particularly in the alveolar septal junctions, corresponding to known location of AT2 cells; AQ5 was distributed in thin bands lining the alveolar walls suggestive of the apical membrane of AT1 cells; αSMA was positive around airways, the known location of smooth muscle cells (SMCs); PECAM-1 was positive within alveolar walls corresponding to microvascular capillaries within alveolar septae. There was no significant cell proliferation during culture under basal conditions. Finally, cell viability studies demonstrated that PCLS can be maintained for up to 1 week in serum-free culture. Conclusion PCLS of human lung parenchyma remain differentiated and viable for up to 7 days in serum-free culture. In future, human PCLS derived from normal and injured regions of lung from the EVLP model may provide a novel means of studying alveolar repair in human lung in vitro.

Effects of antenatal dexamethasone and ambroxol on expression of surfactant protein-B mRNA and ;thyroid transcription factor-1 in pulmonary tissue of premature rats

To explore the effects and mechanism of dexamethasone and ambroxol on expression of surfactant protein （SP）-B mRNA and thyroid transcription factor （TTF）-1 in premature rat lung. Methods Sixteen pregnant Sprague-Dawley rats were randomly divided into four equal groups： two doses of dexamethasone （0.2 mg/kg injected intramuscularly on Day 17 and 18 of pregnancy respectively）;single dose of dexamethasone （0.2 mg/kg injected intramuscularly on Day 18 of pregnancy）;ambroxol group （100 mg/kg injected intraperitoneally on Day 16, 17 and 18 of pregnancy respectively）; and control group （normal saline injected intraperitoneally on Day 16, 17 and 18 of pregnancy respectively）. There were four pregnant rats in each group. All of the fetal rats were taken out on Day 19 of pregnancy as the preterm birth model, and 20 fetal rats from each group were randomly selected. The ratio of body weight to fetal lung weight of newborn rats was calculated. Changes in lung morphology were observed under light microscopy and the ratio of alveoli surface area to alveolar septae surface area was calculated. Expression of TTF-1 protein was determined by immunohistochemistry. Expression of SP-B mRNA was detected by reverse transcriptase-polymerase chain reaction. One-way analysis of variance, Student-Newman-Keuls method and Pearson correlation analysis were applied as statistical methods. Results （1） The ratio of body weight to fetal lung weight was （6.5±0.6）, （7.9±0.8）, （9.5±0.8） and （9.5±0.9） mg/g in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=67.69,P〈0.01）. The ratio of two doses and one dose of dexamethasone group was lower than that of control group （q=17.143 and 9.143, all P〈0.01） and ambroxol group （q=17.143 and 9.143, all P〈0.01）. The ratio of two doses dexamethasone group was lower than that of one dose dexamethasone group （q=8.000, P〈0.01）. （2） The ratio of alveoli surface area to alveolar septae surface area was 2.19±0.15, 1.70±0.18, 1.67±0.13 and 1.08±0.12 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=190.85, P〈0.01）. The ratio of two doses of dexamethasone group, one dose of dexamethasone group and ambroxol group were higher than that of the control group （q=33.639, 18.788 and 17.879, all P〈0.01）. The ratio of two doses dexamethasone group was higher than that of one dose dexamethasone group （q=14.848, P〈0.01）. （3） Expression of TTF-1 protein was 0.311±0.018, 0.224±0.019, 0.196±0.013 and 0.191±0.018 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=211.69,P〈0.01）. TTF-1 protein expression of two doses and one dose of dexamethasone group were higher than that of control group （q=30.000 and 8.250, all P〈0.01） and ambroxol group （q=28.750 and 7.000, all P〈0.01）. TTF-1 protein expression of two doses dexamethasone group was higher than that of one dose dexamethasone group （q=21.750, P〈0.01）. （4） Expression of SP-B mRNA was 1.25±0.13, 1.15±0.12, 1.10±0.10 and 1.01±0.12 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=14.48, P〈0.01）. SP-B mRNA expression of two doses of dexamethasone group, one dose of dexamethasone group and ambroxol group were higher than that of control group （q=9.231, 5.385 and 3.462, all P〈0.01）. SP-B mRNA expression of two doses of dexamethasone group was higher than that of ambroxol group （q=5.769, P〈0.01） and one dose of dexamethasone group （q=3.846, P〈0.01）. （5）TTF-1 expression in two doses of dexamethasone group, one dose of dexamethasone group and control groups was positively correlated with SP-B mRNA expression （r=0.512, 0.597 and 0.449, respectively, all P〈0.05）. Conclusions Ambroxol can accelerate the maturation of fetal lung with minimal adverse effects on fetal lung weight. Ambroxol might be an alternative to dexamethasone to prevent neonatal respiratory distress syndrome. Key words: Dexamethasone; Ambroxol; Pulmonary surfactant-associated protein B; Nuclear proteins; Transcription factors; Infant,premature

Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line.

BackgroundChloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members.ResultsWe analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells.ConclusionsIn a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.

Jaagsiekte sheep retrovirus detected in human lung cancer tissue arrays

BackgroundAdenocarcinoma is the most common type of non-small cell lung cancer and is frequently observed in non-smoking patients. Adenocarcinoma in-situ (formerly referred to as bronchioloalveolar carcinoma) is a subset of lung adenocarcinoma characterized by growth along alveolar septae without evidence of stromal, vascular, or pleural invasion, that disproportionately affects never-smokers, women, and Asians. Adenocarcinoma in-situ is morphologically and histologically similar to a contagious lung neoplasm of sheep called ovine pulmonary adenocarcinoma (OPA). OPA is caused by infection with the exogenous betaretrovirus, jaagsiekte sheep retrovirus (JSRV), whose envelope protein (Env) is a potent oncogene. Several studies have reported that a proportion of human lung adenocarcinomas are immunopositive for an antigen related to the Gag protein of JSRV, however other groups have been unable to verify these observations by PCR.MethodsHere we examine human lung cancer tissue arrays (TA) for evidence of JSRV Env protein and DNA by immunohistochemical staining and PCR, respectively.ResultsOur results reveal that a subset of human lung cancers express an antigen that reacts with a JSRV Env-specific monoclonal antibody in immunohistochemistry and that exogenous JSRV-like env and gag sequences can be amplified from TA tumor samples, albeit inefficiently.ConclusionsWhile a causative role has not been established, these data suggest that a JSRV-like virus might infect humans. With next generation sequencing approaches, a JSRV-like virus in human lung cancers may be identified which could have profound implications for prevention, diagnosis and therapy.

Angiotensin II type 1 and 2 receptors and lymphatic vessels modulate lung remodeling and fibrosis in systemic sclerosis and idiopathic pulmonary fibrosis

OBJECTIVE:To validate the importance of the angiotensin II receptor isotypes and the lymphatic vessels in systemic sclerosis and idiopathic pulmonary fibrosis.METHODS:We examined angiotensin II type 1 and 2 receptors and lymphatic vessels in the pulmonary tissues obtained from open lung biopsies of 30 patients with systemic sclerosis and 28 patients with idiopathic pulmonary fibrosis. Their histologic patterns included cellular and fibrotic non-specific interstitial pneumonia for systemic sclerosis and usual interstitial pneumonia for idiopathic pulmonary fibrosis. We used immunohistochemistry and histomorphometry to evaluate the number of cells in the alveolar septae and the vessels stained by these markers. Survival curves were also used.RESULTS:We found a significantly increased percentage of septal and vessel cells immunostained for the angiotensin type 1 and 2 receptors in the systemic sclerosis and idiopathic pulmonary fibrosis patients compared with the controls. A similar percentage of angiotensin 2 receptor positive vessel cells was observed in fibrotic non-specific interstitial pneumonia and usual interstitial pneumonia. A significantly increased percentage of lymphatic vessels was present in the usual interstitial pneumonia group compared with the non-specific interstitial pneumonia and control groups. A Cox regression analysis showed a high risk of death for the patients with usual interstitial pneumonia and a high percentage of vessel cells immunostained for the angiotensin 2 receptor in the lymphatic vessels.CONCLUSION:We concluded that angiotensin II receptor expression in the lung parenchyma can potentially control organ remodeling and fibrosis, which suggests that strategies aimed at preventing high angiotensin 2 receptor expression may be used as potential therapeutic target in patients with pulmonary systemic sclerosis and idiopathic pulmonary fibrosis.

Impaired Elastin Deposition in Fstl1−/− Lung Allograft under the Renal Capsule

Lung alveolar development in late gestation is a process important to postnatal survival. Follistatin-like 1 (Fstl1) is a matricellular protein of the Bmp antagonist class, which is involved in the differentiation/maturation of alveolar epithelial cells during saccular stage of lung development. This study investigates the role of Fstl1 on elastin deposition in mesenchyme and subsequent secondary septation in the late gestation stage of terminal saccular formation. To this aim, we modified the renal capsule allograft model for lung organ culture by grafting diced E15.5 distal lung underneath the renal capsule of syngeneic host and cultured up to 7 days. The saccular development of the diced lung allografts, as indicated by the morphology, epithelial and vascular developments, occurred in a manner similar to that in utero. Fstl1 deficiency caused atelectatic phenotype companied by impaired epithelial differentiation in D3 Fstl1−/− lung allografts, which is similar to that of E18.5 Fstl1−/− lungs, supporting the role of Fstl1 during saccular stage. Inhibition of Bmp signaling by intraperitoneal injection of dorsomorphin in the host mice rescued the pulmonary atelectasis of D3 Fstl1−/− allografts. Furthermore, a marked reduction in elastin expression and deposition was observed in walls of air sacs of E18.5 Fstl1−/− lungs and at the tips of the developing alveolar septae of D7 Fstl1−/− allografts. Thus, in addition to its role on alveolar epithelium, Fstl1 is crucial for elastin expression and deposition in mesenchyme during lung alveologenesis. Our data demonstrates that the modified renal capsule allograft model for lung organ culture is a robust and efficient technique to increase our understanding of saccular stage of lung development.

Multi-institutional retrospective cohort study of spontaneous pneumothorax

Spontaneous pneumothorax (SP) affects only a subset of patients in a variety of disorders. This multi-institutional study attempted to identify unique clinicopathologic features of spontaneous pneumothorax (SP) in a large cohort. A total of 111 cases from 109 patients were retrieved from 3 institutions over an 11-year period: 27 women, 82 men. 66 were smokers, 16 were non-smokers, 27 unknown. Sixty-six cases (61%) had identifiable disorders (secondary SP). Seven patients had a family history of lung disease: 2 of pneumothorax, 2 of lung cancer, and 1 each of emphysema, COPD, and not further classified. Forty-three cases (32 men, 11 women) were primary. The average age was 28 (men) and 37 (women). 17 smoked, 14 were non-smokers, and 12 unknown. In 26%, a unique recently described entity, pneumothorax-associated fibroblastic lesion (PAFL), was noted. 16% of this subset also had cellular alveolar septae. Abundant intraalveolar macrophages were noted in 12 cases. Five cases of unknown etiology had extensive bullous lung disease not otherwise classified. One patient had unusual cellular areas suggestive of LAM but negative for HMB-45. In conclusion, several distinct morphologic lesions were identified: PAFL, a subset with cellular septae, and a third with numerous intraalveolar macrophages.

An infant with pulmonary interstitial glycogenosis: Clinical improvement is associated with improvement in the pulmonary diffusion capacity

Pulmonary interstitial glycogenosis (PIG) is an idiopathic interstitial lung disease of infants. The underlying pulmonary pathophysiology of PIG has not been well characterized. Herein we report a term-gestation infant who presented with persistent tachypnea and hypoxia. A chest CT scan demonstrated a diffuse ground glass appearance and lung biopsy demonstrated increased alveolar septae cellularity with glycogen-containing cells, consistent with a diagnosis of PIG. At 3 months of age, pulmonary function testing included: pre- and post-bronchodilator forced expiratory flows using the raised-volume technique and the ratio of pulmonary diffusing capacity for carbon monoxide to alveolar volume (DLCO /VA ). He was prescribed 5 days of oral prednisolone (2mg/kg/day) and pulmonary function testing (PFT) was repeated at 5, 13, and 20 months of age. Initial PFTs demonstrated reduced forced vital capacity (FVC: Z-score = -2.36) and an increased ratio of forced expiratory volume in 0.5 sec to FVC (FEV0.5/FVC: Z-score = 1.15) with no significant change following an inhaled bronchodilator. There was also a marked reduction in DLCO /VA (Z-score = -4.74) compared to age-matched controls. Follow-up demonstrated progressive clinical improvement as well as an increase in Z-FVC and normalization of DLCO /VA . Our in vivo physiological findings are consistent with previous reports that symptom resolution correlated with histological thinning of the alveolar septae upon repeat lung biopsy. The restrictive lung disease we observed is consistent with expected reduced compliance of an alveolar interstitial lung process like PIG, whereas the absence of a reduction in FEV0.5/FVC confirms the absence of obstructive airway disease.