Michael A. Moxley

Surfactant protein B deficiency: Antenatal diagnosis and prospective treatment with surfactant replacement†

An infant with a family history of congenital alveolar proteinosis associated with surfactant protein B (SP-B) deficiency was identified when SP-B was not detected in amniotic fluid obtained at 37, 38, and 40 weeks of gestation. Surfactant replacement with commercially available preparations that contained SP-B was begun soon after delivery. Progressive respiratory failure developed despite continued surfactant replacement, corticosteroid therapy, and extracorporeal membrane oxygenation. The infant died at 54 days of age while awaiting lung transplantation. Surfactant extracted from amniotic fluid, bronchoalveolar lavage fluid, and lung tissue had no phosphatidylglycerol; surface tension was 24 dynes/cm (normal, < 10 dynes/cm) and did not decrease with in vitro addition of exogenous SP-B. Pulmonary vascular permeability measured with positron emission tomography was twice normal. At autopsy the alveolar proteinosis pattern was less prominent than that seen in affected siblings. Immunoreactivity of SP-B was absent in type II cells, but numerous foreign body granulomas with central immunoreactivity for SP-B and surfactant protein C were present. We conclude that exogenous surfactant replacement did not normalize surfactant composition, activity, or pulmonary vascular permeability. These findings suggest that endogenous SP-B synthesis is necessary for mature surfactant metabolism and function.

Surfactant Composition and Function in Patients with ABCA3 Mutations

Mutations in the gene encoding the ATP binding cassette transporter member A3 (ABCA3) are associated with fatal surfactant deficiency. ABCA3 lines the limiting membrane of lamellar bodies within alveolar type-II cells, suggesting a role in surfactant metabolism. The objective of this study was to determine the surfactant phospholipid composition and function in patients with mutations in the ABCA3 gene. Bronchoalveolar lavage (BAL) fluid was analyzed from three groups of infants: 1) Infants with ABCA3 mutations, 2) infants with inherited surfactant protein-B deficiency (SP-B), and 3) patients without parenchymal lung disease (CON). Surfactant phospholipid profile was determined using two-dimensional thin-layer chromatography, and surface tension was measured with a pulsating bubble surfactometer. Phosphatidylcholine comprised 41 ± 19% of the total phospholipid in the BAL fluid of the ABCA3 group compared with 78 ± 3% and 68 ± 18%, p = 0.008 and 0.05, of the CON and SP-B groups, respectively. Surface tension was 31.5 ± 9.3 mN/m and was significantly greater than CON but no different from SP-B. We conclude that mutations in ABCA3 are associated with surfactant that is deficient in phosphatidylcholine and has decreased function, suggesting that ABCA3 plays an important role in pulmonary surfactant phospholipid homeostasis.

Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical Application and Future Uses

Chemiluminescence (CL) is an important method for quantification and analysis of various macromolecules. A wide range of CL agents such as luminol, hydrogen peroxide, fluorescein, dioxetanes and derivatives of oxalate, and acridinium dyes are used according to their biological specificity and utility. This review describes the application of luminol chemiluminescence (LCL) in forensic, biomedical, and clinical sciences. LCL is a very useful detection method due to its selectivity, simplicity, low cost, and high sensitivity. LCL has a dynamic range of applications, including quantification and detection of macro and micromolecules such as proteins, carbohydrates, DNA, and RNA. Luminol-based methods are used in environmental monitoring as biosensors, in the pharmaceutical industry for cellular localization and as biological tracers, and in reporter gene-based assays and several other immunoassays. Here, we also provide information about different compounds that may enhance or inhibit the LCL along with the effect of pH and concentration on LCL. This review covers most of the significant information related to the applications of luminol in different fields.

Effect of experimental diabetes and insulin on lipid metabolism in the isolated perfused rat lung

The isolated perfused rat lung was used as a model to study the possible hormonal regulation of lipid metabolism in the mammalian adult lung. Experimental diabetes, whether induced by alloxan or streptozotocin, decreased the incorporation of [U-14C]glucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60-80%. Glucose incorporation into phosphatidylcholine and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the incorporation of glucose into the phosphatidylcholine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidylcholine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mammalian pulmonary surfactant complex.

A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.

Studies on the effects of alloxan and streptozotocin induced diabetes on lipid metabolism in the isolated perfused rat lung

Abstract The effects of experimentally induced diabetes on the conversion of glucose to lipid in the isolated perfused rat lung were examined. Alloxan diabetes and streptozotocin diabetes reduced the incorporation of glucose into the neutral lipid and phospholipid fractions of the lung to a rate less than 40% of that observed in normal animals. This phenomenon appears to be related to insulin deficiency as lungs from diabetic rats treated for one week with insulin were capable of incorporating glucose at a rate comparable to that observed in normal animals. While insulin in vivo altered lipid metabolism in perfused lung, in vitro insulin had no demonstrable effect on lipid metabolism in the perfused lung, an indication that the effects of the hormone may be long term rather than short term. These data indicate that pulmonary lipid metabolism may be regulated by the action of insulin.

Encephalomyocarditis Virus Induces PKR-Independent Mitogen-Activated Protein Kinase Activation in Macrophages

ABSTRACT In this study, we provide evidence that the double-stranded RNA-dependent protein kinase (PKR) is not required for virus-induced expression of inducible nitric oxide synthase (iNOS) or the activation of specific signaling pathways in macrophages. The infection of RAW264.7 cells with encephalomyocarditis virus (EMCV) induces iNOS expression and nitric oxide production, which are unaffected by a dominant-negative mutant of PKR. EMCV infection also activates the mitogen-activated protein kinase, cyclic AMP response element binding protein, and nuclear factor κB (NF-κB) signaling cascades at 15 to 30 min postinfection in PKR+/+ and PKR−/− macrophages. Activation of these signaling cascades does not temporally correlate with PKR activity or the accumulation of EMCV RNA, suggesting that an interaction between a structural component of the virion and the cell surface may activate macrophages. Consistent with this hypothesis, empty EMCV capsids induced comparable levels of iNOS expression, nitrite production, and activation of these signaling cascades to those induced by intact virions. These findings support the hypothesis that virion-host cell interactions are primary mediators of the PKR-independent activation of signaling pathways that participate in the macrophage antiviral response of inflammatory gene expression.

Selective mtDNA mutation accumulation results in β-cell apoptosis and diabetes development

To test the hypothesis that somatic mitochondrial (mt)DNA mutation accumulation predisposes mice to beta-cell loss and diabetes development, transgenic mice expressing a proofreading-deficient mtDNA polymerase-gamma under the control of the rat insulin-1 promoter were generated. At 6 wk of age, mtDNA mutations reached 0.01% (1.05 mutations/10,000 bp) in islets isolated from transgenic mice. This mutational burden is associated with impaired glucose tolerance and a diabetes prevalence of 52% in male transgenic mice. Female transgenic mice maintain slightly elevated fasting glucose levels, mild glucose intolerance, and a diabetes prevalence of 14%. Diabetes in transgenic animals is associated with insulin insufficiency that results from a significant reduction in beta-cell mass. Importantly, apoptosis of beta-cells is increased 7-fold in female and 11-fold in male transgenic mice compared with littermate controls. These results are consistent with a causative role of somatic mtDNA mutation accumulation in the loss of beta-cell mass and diabetes development.

Inhibition of nitric oxide synthase attenuates NNMU-induced alveolar injury in vivo

The purpose of this study was to determine if the acute alveolar injury induced by subcutaneous injections of N-nitroso- N-methylurethane (NNMU) in rats is mediated by nitric oxide (NO ⋅). We show that intraperitoneal injections of the NO ⋅ synthase (NOS) inhibitor N ω-nitro-l-arginine methyl ester (l-NAME) or aminoguanidine significantly attenuate the NNMU-induced alveolar injury as assessed by 1) normalization of the alveolar-arterial O2difference, 2) attenuation of the lowered phospholipid-to-protein ratio in the crude surfactant pellet (CSP), 3) attenuation of the elevated minimal surface tension of the CSP, and 4) attenuation of polymorphonuclear neutrophilic infiltration into the alveolar space. Injections of N ω-nitro-d-arginine methyl ester, the inactive stereoisoform ofl-NAME, did not affect the acute lung injury. Western blot analysis of whole lung homogenates demonstrate an elevated expression of transcriptionally inducible, Ca2+-independent NOS (iNOS) in NNMU-injected rats compared with control saline-injected rats. NOS inhibitors did not affect NNMU-induced iNOS expression. These investigations demonstrate that the inhibition of NOS attenuates NNMU-induced acute lung injury, suggesting a role for NO ⋅ in the progression of acute respiratory distress syndrome.

Effects on Experimental Acute Lung Injury 24 Hours After Exogenous Surfactant Instillation

Subcutaneous injection of N-nitroso-N-methylurethane (NNNMU) produces an acute lung injury mimicking the adult respiratory distress syndrome. NNNMU-injured rats treated with intratracheal Survanta, 100 mg phospholipid/kg body weight, air, or normal saline were observed for 24 h. Twenty-four hours after treatment survival among Survanta-treated rats was significantly greater than for air- and saline-treated rats (9/15 vs. 2/15 and 3/15, respectively). The alveolar-to-arterial O2 gradient was lower in Survanta-treated than in either air- or saline-treated rats during the 24-h period. Analysis of bronchoalveolar lavage fluid revealed a higher phospholipid: protein ratio (1.73 +/- 0.31 Survanta-treated, 0.20 +/- 0.05 air control, and 0.41 +/- 0.17 saline control) and a more normal phospholipid composition among treated than control rats. Minimum dynamic surface tension was significantly lower among treated rats (10.9 +/- 2.9 dyn/cm) than air and saline control rats (36.0 +/- 0.6 and 35.8 +/- 1.0 dyn/com, respectively). In vitro mixing of surfactant with pulmonary edema proteins significantly raised the minimum surface tension of surfactant from a group of Survanta-treated, NNNMU-injured rats (8.7 +/- 3.5 dyn/cm before and 32.0 +/- 0.5 dyn/cm after mixing). Intratracheal Survanta shows a beneficial effect on physiologic parameters and biochemical and functional characteristics of alveolar surfactant for 24 h in rats with NNNMU-induced acute lung injury.