Surfactant replacement therapy--room for improvement.
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Bronchopulmonary Dysplasia↗
Biomedical subjects
R J Mason
Publications and source records attributed to R J Mason.
Contrasting laporoscopic antireflux surgeries: is there a "best"?
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Evaluation Studies as Topic↗
An unusual cause of back pain in a patient with an abdominal aortic aneurysm. A case report.
A case of a 50-year-old man with chronic back pain, an abdominal aortic aneurysm and bony erosion of the lumbar spine due to a chondrosarcoma is reported. This report shows that the lumbar erosion seen in association with an aneurysm is unlikely to be a result of pressure erosion of the spine by the aneurysm, and that an extensive metastatic and septic work-up is required in such patients.
Aged↗
Epitope mapping for monoclonal antibodies identifies functional domains of pulmonary surfactant protein A that interact with lipids.
Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type II cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant SP-As in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a Cys204-Cys218 (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type II cells that is caused by SP-A. From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type II cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type II cells.
Amino Acid Sequence↗
Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction.
Pulmonary surfactant proteins A (SP-A) and D (SP-D) possess similar structure as members of the mammalian C-type lectin superfamily. Both proteins are composed of four characteristic domains which are: 1) an NH2-terminal domain involved in interchain disulfide formation (denoted A1 domain for SP-A or D1 for SP-D); 2) a collagenous domain (denoted A2 or D2); 3) a neck domain (denoted A3 or D3); and 4) a carbohydrate recognition domain (denoted A4 or D4). SP-A specifically binds to dipalmitoylphosphatidylcholine, the major lipid component of surfactant, and can regulate the secretion and recycling of this lipid by alveolar type II cells. SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer), and its role in alveolar lipid metabolism remains to be clarified. To understand the relationship between the structure and the function of both proteins with respect to their interaction with lipids, we expressed recombinant wild type rat SP-D (rSP-D) and chimeric molecules of SP-A and SP-D (A1A2A3D4, A1A2D3D4, and D1D2A3A4) using a baculovirus expression system, and performed lipid binding and aggregation assays. The rSP-D effectively competed with 125I-labeled native rat SP-D in a solid phase binding assay to PI and GlcCer in a manner nearly identical to native SP-D. The rSP-D also bound to PI liposomes with approximately half the affinity of native rat SP-D. Chimera A1A2D3D4 competed with iodinated SP-D in the solid phase binding assay to both PI and GlcCer. This chimera did not bind to dipalmitoylphosphatidylcholine (DPPC) liposomes or induce their aggregation. Chimera A1A2A3D4 did not bind solid phase PI or GlcCer but was equivalent to rSP-D in binding to PI liposomes. This chimera exhibited weak binding to DPPC but failed to aggregate DPPC liposomes. Chimera D1D2A3A4 failed to bind PI and GlcCer and bound weakly to DPPC liposomes but was quite effective at inducing aggregation of DPPC liposomes. These findings demonstrate that the D3 plus D4 domains of SP-D play a role in lipid binding and that the D4 domain is essential for PI binding. Furthermore, the A3 domain of SP-A cannot account for all the lipid binding activity of this protein. In addition, the results implicate the A4 domain of SP-A as an important structural domain in lipid aggregation phenomena.
Amino Acid Sequence↗
Surfactant protein A amino acids Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells.
Pulmonary surfactant protein A (SP-A) is a mammalian lectin that regulates the uptake and secretion of surfactant by alveolar type II cells and is an important component of surfactant complexes. The domains of SP-A which mediate these functions have not been fully mapped. The binding of SP-A to its high affinity receptor on alveolar type II cells is thought to be dependent on a carbohydrate recognition domain (CRD), while the interaction with lipids has been attributed to the hydrophobic neck region of the molecule. To explore the role of the CRD in the interactions of SP-A with type II cells and lipids, we introduced mutations into the cDNA to encode for the substitutions Glu195-->Gln and Arg197-->Asp (SP-Ahyp,Gln195,Asp197) and expressed the mutant protein in insect (Sf9) cells using recombinant baculoviruses. Similar mutations introduced into mannose-binding protein A have been shown to switch the carbohydrate binding specificity from mannose > galactose to the converse. Wild type SP-A produced in Sf9 cells does not contain hydroxyproline (SP-Ahyp), but like rat SP-A it binds to carbohydrate affinity columns, lipids, and the SP-A receptor and is a potent inhibitor of the secretion of surfactant from type II cells (IC50 = 0.5-1.0 micrograms/ml). The SP-Ahyp,Gln195,Asp197 also bound to affinity matrices of galactose-Sepharose and mannose-Sepharose but the indicated mutations rendered the binding at least 100 times more susceptible than SP-Ahyp to competition by free galactose. The SP-Ahyp,Gln195,Asp197 did not compete with rat SP-A for occupancy of its high affinity receptor on type II cells and the mutant protein was 25-50-fold less potent as an inhibitor of the secretion of surfactant from type II cells (IC50 = 26.0 micrograms/ml). Unlike SP-Ahyp, the inhibition of secretion of surfactant by SP-Ahyp,Gln195,Asp197 was reversed by 0.25 M alpha-methylmannoside or galactose. In addition, the SP-Ahyp,Gln195,Asp197 bound avidly to phospholipid but did not aggregate vesicles or augment the uptake of phospholipid into type II cells. We conclude that the binding of SP-A to its receptor and the inhibition of surfactant secretion are critically dependent on the carbohydrate binding specificity of the CRD. Furthermore, phospholipid aggregation and augmentation of phospholipid uptake into type II cells are mediated by the COOH-terminal region of SP-A by a mechanism that is distinct from phospholipid binding.
Animals↗
The structure and function of surfactant protein A. Hydroxyproline- and carbohydrate-deficient mutant proteins.
Pulmonary surfactant protein A (SP-A) regulates the uptake and secretion of phospholipid by alveolar type II cells and is an important component of surfactant lipid aggregates. In an attempt to understand how specific structural domains of SP-A relate to the function of the protein, we used site-directed mutagenesis of the cDNA for SP-A and heterologous expression with baculovirus vectors. Synthesis of the wild type SP-A in insect cells resulted in a form of the protein in which proline residues were not hydroxylated and that is denoted SP-Ahyp. Three mutant SP-As with substitutions in the consensus sequences for glycosylation (SP-Ahyp,glc) to prevent N-linked oligosaccharide attachment at Asn1 and Asn187 were produced, individually and in tandem. The SP-Ahyp was glycosylated at both the Asn1 and Asn187 positions, demonstrated partial sulfhydryl-dependent oligomerization, and formed incomplete oligomers in solution. The SP-Ahyp and SP-Ahyp,glc bound to immobilized carbohydrate and to phospholipid liposomes and partially competed for occupancy of a plasma-membrane receptor for SP-A. The SP-Ahyp and the SP-Ahyp,glc were equally effective inhibitors of the secretion of surfactant lipids from isolated type II cells (IC50 = 0.5 microgram/ml) and aggregated phospholipid liposomes at 20 degrees C. All of the recombinant SP-As demonstrated markedly reduced aggregation of lipid at 37 degrees C. We conclude that the hydroxylation of proline residues is required for perfect oligomerization of SP-A and for thermal stability in the interaction with lipid. Furthermore, recombinant SP-A is able to inhibit the secretion of phospholipid from isolated type II cells and to aggregate lipid vesicles independent of the presence of N-linked carbohydrate or the site of glycosylation.
Animals↗
Gastric emptying and acid-output patterns following a suprapapillary duodenojejunostomy reconstruction with differing lengths of the afferent limb in the baboon.
The importance of afferent limb draining biliopancreatic secretions in Roux-en-Y type reconstructions has never been evaluated experimentally. The effects of the suprapapillary Roux-en-Y duodenojejunostomy operation using a constant duodenal and afferent limb length but a varying afferent limb length was assessed in three groups of five baboons. Gastric emptying, which was assessed preoperatively and postoperatively, was not changed in the baboons with a 12.5-cm-long afferent limb, was significantly delayed in the baboons with a 25-cm-long limb, and was significantly increased in the baboons with a 37.5-cm-long limb. Gastric acid control remained unchanged postoperatively.
Anastomosis, Roux-en-Y↗
Effects of cytokines and endotoxin on lung manganese superoxide dismutase expression and immunohistochemical distribution.
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Animals↗
Lung manganese superoxide dismutase increases during cytokine-mediated protection against pulmonary oxygen toxicity in rats.
Parenteral injection of the cytokines interleukin-1 and tumor necrosis factor, or of endotoxin (lipopolysaccharide), protects rats against lethal pulmonary oxygen toxicity. To determine the potential importance of manganese superoxide dismutase (MnSOD) in this model, we measured MnSOD mRNA and activity in lung. In addition, we confirmed that increases in activities were related to changes in MnSOD protein, which was measured using an enzyme-linked immunosorbentassay (ELISA) technique. After cytokine or endotoxin administration, increases in lung MnSOD mRNA occurred promptly (4 h), with or without hyperoxic exposure. In parallel, lung MnSOD protein and activity were increased after 24 h, and protein levels remained elevated after 52 h. MnSOD activity and protein levels were closely correlated. Neither lung copper-zinc superoxide dismutase (CuZnSOD) mRNA nor activity increased following administration of cytokines. Small increases in CuZnSOD mRNA, which did not exceed those in beta-actin mRNA, occurred early (4 h) after endotoxin, but CuZnSOD activity was unchanged. Immunohistochemistry was used to demonstrate in which cell types the increase in MnSOD protein occurred after cytokine or endotoxin administration. In agreement with ELISA findings, immunoreactive MnSOD appeared to be increased in lung parenchyma, but not in lung neutrophils, 24 h after cytokine or endotoxin treatment. MnSOD was heavily concentrated in alveolar type II cells. However, the numbers of surfactant protein D-positive (type II) cells in lung sections did not appear to be increased after treatment with cytokines or endotoxin. We conclude that early and sustained increases in endogenous MnSOD, but not CuZnSOD or other antioxidant enzymes, are associated with protection of rat lungs against hyperoxic damage by cytokines or endotoxin.
Actins↗
Hepatocyte growth factor is a growth factor for rat alveolar type II cells.
Proliferation of alveolar type II cells is thought to be critical for restoration of gas exchange units after diffuse alveolar damage. However, the factors that regulate type II cell proliferation are not well understood. Hepatocyte growth factor (HGF) is a potentially important mitogen because it causes epithelial cells but not fibroblasts to proliferate and is found in the lung. We used rat alveolar type II cells in primary culture to demonstrate that HGF stimulates DNA synthesis in a concentration-dependent manner. The half maximal effect on stimulation of thymidine incorporation was less than 1 ng/ml. By autoradiography, HGF increased nuclear labeling from 1.3% of type II cells with medium alone to 9.4% with 5 ng/ml HGF. During this time, HGF modestly increased cell number in comparison to control media. However, in an assay of colony formation in low-density cultures, HGF did not consistently increase colony formation by alveolar type II cells and was less effective than acidic fibroblast growth factor or bronchoalveolar lavage fluid in this assay. The receptor for HGF (c-met proto-oncogene) was expressed in rat type II cells and whole lung but not in macrophages. In contrast, the mRNA for HGF was detected in rat macrophages and lung but not in type II cells. However, HGF message was not detected in human alveolar macrophages under conditions in which the HGF message was detected in rat alveolar macrophages and in human fibroblasts. Hence, HGF is a potential paracrine growth factor for alveolar type II cells, but there may be important species differences in the relative level of expression.
Animals↗
Agarose infiltration improves morphology of cryostat sections of lung.
BACKGROUND: Optimal morphology and immunohistochemistry of the lung requires the organ to remain fully distended. When the lung is prepared for frozen sections, especially during the stage of cryoprotection, liquid fixatives leak out and the lung tends to collapse. We sought to develop a new technique to improve the morphology of frozen sections of the lung while facilitating tissue handling and immunoreactivity. EXPERIMENTAL DESIGN: Human, fetal sheep and adult rat lungs were distended with 1% low temperature melting agarose and vessels were perfused with a 1% paraformaldehyde solution. RESULTS: The agarose infiltration permitted easy handling of the lung tissue, and maintained lung architecture during cryoprotection. Agarose infiltration and paraformaldehyde fixation provided excellent morphology, that was comparable to paraffin-embedded tissue. The structural preservation was especially noticeable with human lung that remained well distended with the agarose technique. Because of the mild fixation and good morphology, precise localization of antigenic sites was possible for the surfactant proteins SP-A, SP-C and SP-D, cytokeratin, and alveolar macrophage markers. CONCLUSIONS: This procedure can improve special fixation and embedding protocols for lung immunocytochemistry and immunofluorescence.
Animals↗
Medial meniscal cyst of the knee simulating a solitary bone lesion. A case report and review of the literature.
Meniscal cysts are common lesions, but extensive bony changes arising from these cysts are exceedingly rare. These changes are usually erosive defects about the tibia. Dull pain with an associated mass are classic findings. Magnetic resonance imaging is useful in making the diagnosis and arthroscopic management yields excellent results. A 47-year-old man developed a symptomatic medial meniscal cyst that invaded the medial femoral condyle simulating a solitary bone lesion. Magnetic resonance imaging revealed the contiguous nature of the cyst with the meniscus. Arthrotomy with cyst excision was required in this case and led to the amelioration of symptoms.
Cysts↗
'Bile' in the oesophagus.
Acid gastro-oesophageal reflux occurs when the lower oesophageal sphincter is incompetent, but oesophagitis caused by reflux of duodenal content implies incompetence of both the pyloric and gastro-oesophageal sphincters. The term 'alkaline' reflux oesophagitis was coined long before objective analysis was made of bile in the stomach and oesophagus, and well before pH monitoring was introduced. Surgical procedures to divert bile from the stomach and oesophagus were developed on a clinical basis and gave encouraging results in the management of peptic oesophageal stricture. Alkaline oesophagitis is well recognized after gastric surgery and the entity 'primary pathological duodenogastric reflux', although contested by some, attracts growing support. Recent evidence suggests that the complications of Barrett's oesophagus may be related to duodenogastro-oesophageal reflux. Probes designed to measure gastric and oesophageal bile salts have recently been developed and may give more information in the future.
Bile Acids and Salts↗
The effect of fibronectin on cytoskeleton structure and transepithelial resistance of alveolar type II cells in primary culture.
The cytoskeleton of alveolar type II cells on different matrices has been examined, and the bioelectric properties of these cells grown as monolayers in primary culture has be measured using Ussing-type chambers, to determine whether the extracellular matrix affects the cytoskeletal organization of alveolar type II cells and whether any such interactions influence their physiological functions. Alveolar type II cells cultured on a fibronectin substratum spread slowly over a 6-day period to produce cells of extremely large diameter. The cytoskeletal structure of these cells was characterized by a more marked accumulation of large bundles of actin and a finer network of keratin than cells grown on a collagen substratum. The transepithelial resistances of monolayers grown on a fibronectin substratum were much higher than those on a collagen substratum. These results indicate that alveolar type II cells cultured on fibronectin can form tighter, better organized and more polarized monolayers in primary culture, which suggests that fibronectin may have a physiologically important role in the maintenance of the alveolar wall.
Animals↗
The development of a strategy for the implementation of automation in a bioanalytical laboratory.
Laboratory automation is equipment, instrumentation, software and techniques that are classified into four groups: instrument automation; communications; data to information conversion; and information management. This new definition is necessary to understand the role that automation can play in achieving the aims and objectives of a laboratory within its organization. To undertake automation projects effectively, a laboratory automation strategy is outlined which requires an intimate knowledge of an organization and the target environment to implement individual automation projects.
Automation↗
Motility differences between long-segment and short-segment Barrett's esophagus.
Barrett's esophagus has been described as a motility disorder of the esophagus, but the issue of whether or not the disorder precedes the columnar lining has been debated. Motility records of 70 patients with histologically proven Barrett's esophagus were analyzed. A comparison of the swallow responses was made between those patients who had a limited Barrett's segment (3 to 5 cm, n = 28) as opposed to those patients who had an extensive Barrett's segment (greater than 5 cm, n = 42). Patients with an extensive segment had a significantly lower amplitude (p < 0.01) of esophageal contraction and a significantly longer duration (p < 0.01) of contraction compared with patients with a limited segment. These differences were most marked in the distal esophageal segments. There was a progressive deterioration of esophageal motility with advanced disease.
Adult↗
Proliferation of rat alveolar epithelial cells in low density primary culture.
Alveolar type II cells proliferate to restore the alveolar epithelium after lung injury and differentiate into type I epithelial cells. A variety of factors promote rat type II cell DNA synthesis in vitro; however, only low levels of proliferation occur when type II cells are cultured at high density. We plated type II cells at low density to determine if those growth factors that stimulate thymidine incorporation also stimulate low density proliferation. Type II cells were plated at 1 x 10(3) cells/cm2 in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum, cholera toxin, insulin, epidermal growth factor, acidic fibroblast growth factor (aFGF), and concentrated bronchoalveolar lavage fluid from normal rats. By 7 days, numerous colonies had grown out that exhibited an epithelial morphology and stained positively for cytokeratin. The cell number at day 7 in the presence of the combined factors was 5.9 x 10(3) (+/- 0.6 x 10(3)) cells/cm2 (n = 4). There was no colony formation in the absence of fetal bovine serum. The addition of linoleic acid to serum-free medium containing all the growth supplements was found to partially restore colony formation. When aFGF or lavage fluid was omitted from the culture medium, colony formation was dramatically reduced. The colonies lacked characteristics of differentiated type II cells, which was anticipated since these cells were cultured on tissue culture plastic. To see if these cells could express differentiated functions, we maintained the colonies under growth conditions, removed them from the plastic substratum, and then replated them on EHS matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗