Regulation of the beta 2 subunit chain of laminin in developing rabbit fetal lung tissue

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The beta 2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the beta 2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin beta 2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin beta 2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin beta 2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin beta 2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin beta 2 protein induction observed in fetal lung tissue, laminin beta 2 mRNA levels were maximal late in gestation. Utilizing a laminin beta 2 chain cRNA probe and in situ hybridization, we detected laminin beta 2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin beta 2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin beta 2 chain may be involved in alveolar epithelial cell differentiation.     

Temporal pattern of accelerated lung growth after tracheal occlusion in the fetal rabbit

Tracheal occlusion in utero is a potent stimulus of fetal lung growth. We describe the early growth mechanics of fetal lungs and type II pneumocytes after tracheal ligation (TL). Fetal rabbits underwent TL at 24 days gestational age (DGA; late pseudoglandular stage; term = 31 to 33 days) and were sacrificed at time intervals ranging from 1 to 5 days after TL. Lung growth was measured by stereological volumetry and bromodeoxyuridine (BrdU) pulse labeling. Pneumocyte II population kinetics were analyzed using a combination of anti-surfactant protein A and BrdU immunohistochemistry and computer-assisted morphometry. Nonoperated littermates served as controls. TL resulted in dramatically enhanced lung growth (lung weight/body weight was 5.00 +/- 0.81% in TL versus 2.52 +/- 0.13% in controls at 29 DGA; P < 0.001, unpaired Student's t-test). Post-TL lung growth was characterized by a 3-day lag-phase typified by relative stagnation of growth, followed by distension of airspaces, increased cell proliferation, and accelerated architectural and cellular maturation by postligation days 4 and 5. During the proliferation phase, the replicative activity of type II cells was markedly increased (type II cell BrdU labeling index was 10.0 +/- 4.1% in TL versus 1.1 +/- 0.3% for controls at 29 DGA; P < 0.02), but their numerical density decreased (3.0 +/- 0.5 x 10(-3)/microm2 in TL versus 4.5 +/- 0.3 x 10(-3)/microm2 in controls at 29 DGA; P < 0.02), suggesting accelerated terminal differentiation to type I cells. In conclusion, post-TL lung development is characterized by a well defined temporal pattern of lung growth and maturation. The rabbit model lends itself well to study the regulatory mechanisms underlying accelerated fetal lung growth after TL.     

Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses

Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.     

Pulmonary lesions in experimental ophidian paramyxovirus pneumonia of Aruba Island rattlesnakes, Crotalus unicolor

Histologic and ultrastructural changes were observed in the respiratory portions of lung in five 29-40-month-old Aruba Island rattlesnakes, Crotalus unicolor, that were inoculated with an Aruba Island Rattlesnake virus (AIV) strain of ophidian paramyxovirus (OPMV) isolated from an Aruba Island rattlesnake. Lungs from one non-infected and three mock-infected Aruba Island rattlesnakes were examined also. From 4 to 22 days following intratracheal inoculation, progressive microscopic changes were seen in the lung. Initially, increased numbers of heterophils were observed in the interstitium followed by proliferation and vacuolation of epithelial cells lining faveoli. The changes appeared to progress from cranial to caudal portions of the respiratory lung following inoculation. Beginning at 4 days postinoculation, viral antigen was demonstrated in epithelial cells lining faveoli with an immunofluorescent technique using a rabbit anti-AIV polyclonal antibody. Electron microscopy revealed loss of type I cells, hyperplasia of type II cells, and interstitial infiltrates of heterophils and mononuclear cells. Viral nucleocapsid material was seen within the cytoplasm and mature virus was seen budding from cytoplasmic membranes of infected type I and type II cells from 8 to 19 days after infection. A virus consistent with AIV was isolated from lung tissues of infected rattlesnakes, thus fulfilling Koch's postulates.     

Compensatory occurrence of IV2Fucalpha,II3NeuAcalpha-Gg4Cer and human fetal antigen Lc4Cer in small cell carcinomas of the human lung

By means of a thin-layer chromatography immunostaining procedure involving a human monoclonal anti-Lc4Cer antibody, which was established by hybridizing murine myeloma cells and human lymphocytes from a cancer patient, Lc4Cer was proven to be a fetal antigen of human lung and to be a cancer-related antigen in small cell carcinomas of human lung, but not of other lung cancers, i.e., large cell carcinomas, adenocarcinomas, and squamous carcinomas. With the simultaneous detection of IV2Fuc alpha,II3NeuAc alpha-Gg4Cer with rabbit anti-IV2Fuc alpha,II3NeuAc alpha-Gg4Cer antiserum, the expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer was found to be compensatory and, consequently, small cell lung carcinomas could be classified into Lc4Cer- and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer-expressing types, L-SCLC and F-SCLC, respectively, which were detected in four and 27 of 31 patients' tissues and in one and three of four nude mouse-transplanted small cell lung carcinoma tissues, respectively. The compensatory expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer in small cell carcinomas indicated that different metabolic pathways for glycosphingolipids were activated to give the distinct glycosphingolipid compositions in the two types of small cell lung carcinomas.     

Characterization of stromal progenitor cells enriched by flow cytometry

The progenitors for cells of bone, cartilage, fat, and muscle are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well-characterized. In this study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labeling index with [3H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies show that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods. These cells may be useful for regeneration of stromal tissues.     

Prenatal medicine: development, current status and future perspectives

Prenatal diagnosis and therapy are based mainly on the progress of diagnostic ultrasound and laboratory methods in genetics. There is a general tendency to replace second trimester fetal diagnoses by first trimester approaches. Transvaginal sonography and chorionic villus sampling in particular have been proven helpful in this context. The aim of all prenatal diagnostic measures is fetal therapy in time to prevent untreatable abnormalities. There has been some progress in this area; in particular, the application of stem cells for the correction of single cell diseases seems to be promising. Because all prenatal interventions involve a risk to the mother and especially the fetus, there is a concentrated effort to develop non-invasive screening or diagnostic methods. Extensive work on the isolation of fetal cells from the maternal circulation has revealed that such cells are present physiologically in pregnancy, but further trials need to show whether this method is safe enough for routine diagnostic use.     

Alveolar stem cell transduction by an adeno-associated viral vector

In inherited disorders such as surfactant protein deficiencies or cystic fibrosis (CF), where lung damage develops progressively after birth, gene replacement is best accomplished in the neonatal period. We use the adeno-associated virus (AAV) as a vector for gene transfer in the newborn rabbit lung where stem cells are activated for lung growth and differentiation. AAV-mediated gene transfer as assayed by lacZ gene expression occurred preferentially in alveoli in the alveolar epithelial progenitor cell, the type II cell, and in the large airway tracheobronchial basal and ciliated cells. Cell proliferation was confirmed by 5-bromo-deoxyuridine (BRDU) labeling in regions undergoing alveolarization and airway branch points. Regions of cell proliferation coincided with areas of significant lacZ expression. Thus, dividing and differentiating cells can be targeted by AAVlacZ delivery to newborn lung.     

Purification of a cell-surface receptor for surfactant protein A

In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens.     

Stimulation of phospholipase activity and prostaglandin biosynthesis by melittin in cell culture and in vivo

Melittin, a membrane-active peptide of bee venom, as well as synthetic melittin, stimulated the biosynthesis of prostaglandins by mouse transformed fibroblasts (MC5-5), human fibroblasts (D550), rabbit aorta endothelial cells (CLO), rat lung type II alveolar pneumocytes (L-2) and rabbit smooth muscle cells (R-I). The melittin peptides also stimulated the release of arachidonic acid from the cellular phospholipids of MC5-5 cells. The stimulated prostaglandin biosynthesis by MC5-5 cells was inhibited by indomethacin and dexamethasone. Dexamethasone inhibited also the release of arachidonic acid by MC5-5 cells. In mice, intraperitoneal inoculation of melittin increased 13,14-dihydro-15-keto-PGE2 levels in peripheral blood. Prior injections of the mice with indomethacin prevented the melittin-induced increase in this PGE2 metabolite.     

Oxidant injury to the alveolar epithelium: biochemical and pharmacologic studies

This multifaceted study involved a combined biochemical and cellular analysis of oxidant metabolism by a lung cell at risk from injury by endogenous and environmental oxidants, the pulmonary alveolar type II epithelial cell. Within the framework of this study, a method was developed for effectively delivering antioxidant enzymes and alpha-tocopherol to the intracellular compartment of alveolar epithelial cells. Alveolar type II cells are key sources of pulmonary surfactant phospholipids and apoproteins and serve as progenitors of type I alveolar epithelium, thus playing an important role in the re-epithelialization of the lung alveolus after exposure to pulmonary oxidants. The type I and II pulmonary epithelium also play an essential collaborative role in maintaining the integrity of the air-blood barrier of the lung. Because of these critical properties of the alveolar epithelium and their recognized sensitivity to oxidant stress derived from diverse sources, such as activated inflammatory cells, hyperoxia, the environmental oxidants and nitrogen dioxide, and surgical procedures, such as cardiopulmonary bypass and lung transplantation, we endeavored to understand more about the oxidant metabolism and antioxidant pharmacology of these cells. In our experiments, we made the observation that loss of differentiated oxidant generation and antioxidant properties of type II cells occurs very rapidly in vitro. For example, we observed a 50% to 75% reduction in the specific activities of type II cell superoxide dismutase, catalase, and glutathione peroxidase, all critical scavengers of cell superoxide and hydrogen peroxide and key enzymes in the attenuation of hydroxyl radical formation. Although the differentiated characteristics of the type II cell antioxidant defenses changed in vitro, they may have become more reflective of type I alveolar epithelial cells. The type I cell is the most vulnerable for oxidant damage in the alveolus because of its large surface area and the possibility of a reduced antioxidant capacity compared to type II alveolar epithelium. In spite of this limitation, we were able to culture type II cells and study their adaptive and toxic responses to exogenously administered oxidant stress. We also observed that a significant source of self-generated oxidants in type II cells was the enzyme xanthine oxidase. Normal rates of oxidant production by this enzyme had an inhibitory effect on incorporation of biosynthetic precursors into surfactant phospholipids; these effects were eliminated by the xanthine oxidase inhibitor, allopurinol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homology modelling by distance geometry

The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Type I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.     

Analysis of forensic psychiatric opinions issued in Warsaw in 1968 during legal proceedings for deprivation of civil rights

The isolation and characterization of type III collagen from adult human lung parenchyma are described. The identity of this molecule as type III collagen has been established on the basis of (a) demonstration of intramolecular disulfide cross-links in the helical portion of the molecule, (b) amino acid analysis characteristic for type III collagen, and (c) composition and size of isolated cyanogen bromide peptides alpha1(III)-CB3, alpha1(III)-CB5, and alpha1(III)-CB8. The molecular weight of lung alpha1(III) was determined as 93 000 by Agarose chromatography, but its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels was slower than that of type I alpha chains which also have a molecular weight of 93 000.     

Histological and ultrastructural observations on the development of the lung of the fetal pig

Lungs of fetal pigs having gestational ages ranging from 80 to 115 days were examined histologically and by electron microscopy. At 80 days bronchial epithelium was ciliated but bronchiolar cells were not and bronchial mucosal glands were absent. Peripheral regions consisted predominantly of mesenchymal tissue with glandular alveoli. 92 days marked the transition from the immature to the more mature lung type. Bronchial glands appeared and began to grow from the epithelium into the lamina propria, bronchiolar epithelial cells acquired cilia, and alveoli were becoming irregular in shape and had thinner interalveolar septa. Close contact between capillaries and alveolar epithelium established the blood-air barrier at many points. Differentiation of alveolar epithelium into types I and II pneumonocytes occurred at this stage and lamellated osmiophilic inclusion bodies were present in type II cells for the first time. The number of lamellated bodies increased progressively to term at 115 days.     

Recognition of normal, neoplastic, and fetal airway epithelial cell membranes by two monoclonal antibodies

The reactivity of two rat monoclonal antibodies was studied. These antibodies, A2R and A2C, bind a 32 kDa alveolar type II cell membrane receptor for surfactant protein A. A2R and A2C also bind apical cell membranes of ciliated and nonciliated cells of the conducting airways. Because this reactivity suggested possible utility in targeting those cells for therapeutic gene transfer, the binding activity of these two antibodies was examined in human tissues. In conducting airways, A2R and A2C bound apical epithelial cell membranes throughout the embryologic period studied: from 15 weeks of gestation, through maturity. Reactivity was more restricted to ciliated cells of the airways as maturation progressed. In the peripheral lung, A2C and A2R only bound most cells in the early developing lung, but mainly type II cells in mature lungs. Other normal tissues recognized by these antibodies included crypt lining cells of the adult and fetal stomach, large bile duct epithelium, and pancreatic acinar cells. All of these cells derive from embryonic foregut endoderm. Other normal tissues, both of endodermal and nonendodermal origin, were negative. Pulmonary carcinomas were studied. A2C and A2R recognized all non-small cell carcinomas of the lung tested. In contrast, none of the small cell carcinomas or carcinoid tumors of the lung were recognized by these antibodies. The function of p32 in these diverse cell types is not clear, but whatever its role in these tissues, antibodies versus p32 may potentially be used to target gene or drug therapy to the normal or malignant cells they recognize.     

Boson analyses in the Ge isotopes

The renin-angiotensin system is activated during vascular development and injury. Furthermore, angiotensin II (Ang II) is a comitogen for fetal mesangial cells in vitro and it may be important in vascular smooth cell growth in disease states. Since fibronectin is an important extracellular matrix protein for vascular development and it too is overexpressed in the mesangium of diseased glomeruli, we explored the interrelationship of fibronectin and Ang II in fetal mesangial cell growth. In human fetal kidney, Ang II type 2 receptors (AT2) were detected in abundance by ex vivo autoradiography. When mesangial cells were isolated from fetal kidney and grown in culture, Ang II type 1 receptors (AT1) were also detected. To explore the mitogenic properties Ang II and fibronectin as well as the effects of Ang II on fibronectin metabolism, studies were performed in vitro, isolated from the potentially confounding variables of hemodynamic influence and circulating growth factors and cytokines. In vitro, mesangial cells expressed a single class of AT1 receptors that were not altered by growth on various substrates. Ang II (10(-7) M) significantly increased thymidine incorporation by confluent human fetal mesangial cells (twofold). When subconfluent, Ang II-stimulated proliferation was greater (fourfold). Ang II significantly increased cell-associated and secreted fibronectin as determined by immunoprecipitation at concentrations that also stimulate mitogenesis. Both of these Ang II-mediated responses were inhibited by the AT1 receptor antagonist DuP-753 (10(-5) M) but not by AT2 receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)