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Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) differ for triggers, mode of start, associated symptoms, evolution, and biochemical traits. Therefore, serious attempts are underway to partition them into subgroups useful for a personalized medicine approach to the disease. Here, we investigated clinical and biochemical traits in 40 ME/CFS patients and 40 sex- and age-matched healthy controls. Particularly, we analyzed serum levels of some cytokines, Fatty Acid Binding Protein 2 (FAPB-2), tryptophan, and some of its metabolites via serotonin and kynurenine. ME/CFS patients were heterogeneous for genetic background, trigger, start mode, symptoms, and evolution. ME/CFS patients had higher levels of IL-17A (p = 0.018), FABP-2 (p = 0.002), and 3-hydroxykynurenine (p = 0.037) and lower levels of kynurenine (p = 0.012) and serotonin (p = 0.045) than controls. Changes in kynurenine and 3-hydroxykynurenine were associated with increased kynurenic acid/kynurenine and 3-hydroxykynurenine/kynurenine ratios, indirect measures of kynurenine aminotransferases and kynurenine 3-monooxygenase enzymatic activities, respectively. No correlation was found among cytokines, FABP-2, and tryptophan metabolites, suggesting that inflammation, anomalies of the intestinal barrier, and changes of tryptophan metabolism may be independently associated with the pathogenesis of the disease. Interestingly, patients with the start of the disease after infection showed lower levels of kynurenine (p = 0.034) than those not starting after an infection. Changes in tryptophan metabolites and increased IL-17A levels in ME/CFS could both be compatible with anomalies in the sphere of energy metabolism. Overall, clinical traits together with serum biomarkers related to inflammation, intestine function, and tryptophan metabolism deserve to be further considered for the development of personalized medicine strategies for ME/CFS.

Surfactant protein B (SP-B) plays a key role in surfactant homeostasis affecting its biophysical properties and physiological function. Recently, a method to measure SP-B amount and kinetics from tracheal aspirates (TAs) became available. The main objective of this study was to improve the critical steps of the procedure to obtain a better SP-B sensitivity. We administered a 24 h continuous infusion of 1 mg/kg/h of 113 C-leucine to ten newborn infants. SP-B was isolated from serial TAs and its fractional synthesis rate, secretion time, peak time and half life were derived from 13 C enrichment curves obtained by gas chromatography mass spectrometry. SP-B amount in TAs was also assessed. During the extraction step, acidification and organic solvent ratio optimization doubled the recovery of SP-B from TAs, so did the elongation of the propylation time (from 20 min to 1 h) with enhanced leucine derivatization yield. Measurement of 13 C leucine enrichments, and therefore all SP-B kinetics parameters, were successfully calculated in all TAs samples due to the increase of SP-B yield. SP-B amount was 0.29 (0.16–0.41) % of total phospholipids with a minimum value of 0.08% belonging to one of the respiratory distress syndrome (RDS) patients. In conclusion, this new procedure enables accurate determination of SP-B kinetics even in the presence of low protein amount like in preterm RDS patients. Copyright © 2012 John Wiley & Sons, Ltd.

Disaturated-phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) are respectively the first and the third most abundant phospholipid in human alveolar surfactant. Their concentration decreases in airway surfactant of adults and infants with respiratory distress syndrome and cystic fibrosis. In this study, we used mass spectrometry (IRMS) to investigate the turnover of DSPC and PG in tracheal aspirates (TA) obtained from infants with normal or diseased lungs. We studied eight infants requiring mechanical ventilation: two with no lung disease, four with diaphragmatic hernia, one with ATP-binding cassette sub-family A member 3 heterozygote mutation and one with sepsis. Patients received deuterated water for 48 h as metabolic precursors of palmitate-DSPC and palmitate-PG. Serial TAs were obtained every 6 h for five days or until extubation. DSPC and PG were isolated from TA by column and high-performance thin layer chromatography. Deuterium enrichments of palmitate-DSPC and PG residues were measured by IRMS coupled with a gas chromatographer. Median secretion time (ST), peak time (PT) and fractional synthesis rate (FSR) were 3.7 [0.9– 13.4] h, 71.0 [52.2 – 85.2] h and 6.6 [6.3 – 11.1] %/day for DSPC and 19.3 [6.4 – 22.8] h, 49.0 [33.0 – 52.5] h and 5.8 [4.8 – 10.9] %/day for PG. This study shows that it is feasible to use deuterium derived from body water to trace simultaneously airway surfactant DSPC and PG in humans. When compared within the same patient, DSPC and PG had similar fractional synthesis rates, but PG had a shorter PT, suggesting differences in the life cycle of these essential surfactant components. Copyright © 2011 John Wiley & Sons, Ltd.

Amiodarone (AMI) is a potent antiarrhythmic agent; however, its clinical use is limited due to numerous side effects. In order to investigate the structure--cytotoxicity relationship, AMI analogues were synthesized, and then, using rabbit alveolar macrophages, were tested for viability and for the ability to interfere with the degradation of surfactant protein A (SP-A) and with the accumulation of an acidotropic dye. Our data revealed that modification of the diethylamino-beta-ethoxy group of the AMI molecule may affect viability, the ability to degrade SP-A and vacuolation differently. In particular, PIPAM (2d), an analogue with a piperidyl moiety, acts toward the cells in a similar manner to AMI, but is less toxic. Thus, it would be possible to reduce the cytotoxicity of AMI by modifying its chemical structure.

Surfactant protein C (SP-C) is deemed as the surfactant protein most specifically expressed in type II alveolar epithelial cells and plays an important role in surfactant function. SP-C turnover in humans and its meaning in the clinical context have never been approached. In this study, we used mass spectrometry to investigate SP-C turnover in humans. We studied four infants and eight adults requiring mechanical ventilation. All patients had no lung disease. Patients received a 24-h continuous infusion of (13)C-leucine as precursor of SP-C, and serial tracheal aspirates and plasma samples were obtained every 6 h till 48 h. SP-C was isolated from tracheal aspirates by sorbent-phase chromatography. (13)C-leucine SP-C enrichment could be successfully measured in three infant and in four adult samples by using mass spectrometry coupled with a gas chromatographer. Median SP-C fractional synthesis rate, secretion time, and peak time were 15.7 (14.1-27.5)%/day, 6.0 (4.7-11.5) h, and 24 (20-27) h. In conclusion, this study shows that it is feasible to accurately determine SP-C turnover in humans by stable isotopes.

Graphical abstract Amiodarone, having basic pKa and high water solubility at acidic pH, accumulates within late endocytic compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking and inducing a Niemann-Pick C-like phenotype., Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control.

Backgroundα1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series ofin vitroexperiments aimed at investigating the nature and consequences of such an interaction.Methods and resultsAt an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.ConclusionWe conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.

Amiodarone, an antiarrhythmic drug toxic toward the lung, is metabolized through sequential modifications of the diethylaminoethoxy group to mono- N-desethylamiodarone (MDEA), di- N-desethylamiodarone (DDEA), and amiodarone-EtOH (B2-O-EtOH), whose effects on lung cells are unclear. To clarify this, we exposed rabbit alveolar macrophages to analogs with different modifications of the diethylaminoethoxy group and then searched for biochemical signs of cell damage, formation of vacuoles and inclusion bodies, and interference with the degradation of surfactant protein A, used as a tracer of the endocytic pathway. The substances studied included MDEA, DDEA, and B2-O-EtOH, analogs with different modifications of the diethylaminoethoxy group, fragments of the amiodarone molecule, and the antiarrhythmic agents dronedarone (SR-33589) and KB-130015. We found the following: 1) MDEA, DDEA, and B2-O-EtOH rank in order of decreasing toxicity toward alveolar macrophages, indicating that dealkylation and deamination of the diethylaminoethoxy group represent important mechanisms of detoxification; 2) dronedarone has greater, and KB-130015 has smaller, toxicity than amiodarone toward alveolar macrophages; and 3) the benzofuran moiety, which is toxic to liver cells, is not directly toxic toward alveolar macrophages.

In animal models of congenital diaphragmatic hernia (CDH), surfactant deficiency contributes to the pathophysiology of the disease; however, information on CDH in humans is limited. We compared surfactant disaturated phosphatidylcholine (DSPC) synthesis and metabolism, by stable isotope technology, in newborn infants with CDH and in control subjects. DSPC amount, total proteins, and surfactant protein-A (SP-A) from tracheal aspirates were also measured. DSPC and SP-A were significantly lower in 14 infants with CDH than in the eight control subjects. Mean DSPC was 2.3 +/- 1.3 mg/ml of epithelial lining fluid (ELF) in infants with CDH and 4.6 +/- 1.5 mg/ml of ELF in control subjects (p = 0.001). Mean SP-A in infants with CDH and in control subjects was 16.2 +/- 9.3 and 61.2 +/- 30.6 microg/ml of ELF, respectively (p = 0.03). DSPC kinetics was measured in 12 of 14 infants with CDH and in 5 of 8 control subjects. Secretion time was 8.3 +/- 5.5 and 8.5 +/- 2.5 hours and peak time 51.9 +/- 15.2 and 51 +/- 13 hours in infants with CDH and in control subjects, respectively. Fractional synthesis rate was not different for infants with CDH and control subjects (p = 0.4). In conclusion, surfactant DSPC synthesis and kinetics were not significantly deranged in infants with CDH compared with control subjects. Other factors, such as lower surface area or increased DSPC catabolism, may contribute to surfactant pool alteration in CDH.

A 51-year-old man with chronic alcoholic liver disease developed a severe haemolytic anaemia characterized by the presence of circulating burr-shaped cells (echinocytes). Several transfusions of packed red cells were ineffective in raising the haemoglobin concentration, showing that the abnormality was acquired by the transfused cells. Liver biopsies revealed haemochromatosis. Haematological parameters normalized four months after the patient stopped drinking alcohol, but burr cells were still present and erythrocyte life-span was still markedly shortened at one year follow-up. Since serum cholesterol, HDL-cholesterol, and Apo-AI and Apo-B lipoproteins were considerably decreased, the lipid composition of the red cell membrane was studied. Findings showed that echinocytosis occurred with no change in membrane cholesterol content, nor in cholesterol:phospholipid ratio, but with an alteration in the phosphatidylserine and phosphatidylinositol concentrations. While haemochromatosis was most likely the cause of the erythrocyte anomaly, alcohol intake was probably responsible for the acute onset of haemolytic anaemia with effects directly on the erythrocyte membrane as well as mediated by the progressive hepatic injury, with alterations in the plasma and successively in the intramembrane lipid composition.

Surfactant subtypes have been identified by gradient centrifugation and characterized by differences in protein composition, vesicle size, and surface activity. The presence of surfactant proteins in each subtype is thought to be critical to the formation of aggregates and their function (Baritussio, Am J Physiol 1994;266:L436). To evaluate whether synthetic surfactant peptides interact with lipids in a manner comparable to native surfactant proteins, we prepared surfactant vesicles containing synthetic 125I-SP-B1-78 and phospholipids (DPPC:PG:Palmitic acid, 7:2:1) [PL], with and without palmitoylated synthetic SP-C1-34. We separated the aggregates by continuous sucrose gradient (0.1-0.8 M; 100,000 g for 48 h), collecting 50 fractions, and analyzed the PL and peptide profile. The PL peak appeared in fraction 17 and > 80% of PL was recovered in fractions 15-17, at a density of 1.07 g/mL, irrespective of the presence of SP-C. In the SP-B/PL aggregates, SP-B peaked at 51% in fraction 15 and > 95% of SP-B appeared in fractions 11-17. In the SP-B&SP-C/PL aggregates, 17% of the SP-B was detected at the bottom of the gradient with

To estimate the turnover time of alveolar surfactant in New Zealand white rabbits, we injected [9,10- 3 H]palmitic acid and [2- 14 C]glycerol intravenously. From 1–48 h after injection, we killed the animals, lavaged the lungs for alveolar surfactant with saline, and isolated the lamellar bodies by homogenization and sucrose density gradient centrifugation. Lamellar bodies and alveolar surfactant had comparable phospholipid composition and surface activity. Lamellar bodies contained little DNA, no mitochondrial enzyme activity and less than 5% contaminating phospholipids from microsomal and Golgi-enriched fractions. We measured radioactivity of phosphatidylcholine, saturated phosphatidylcholine and phosphatidylglycerol for each isotope in lamellar bodies and surfactant at each time point. The plot of the integral with respect to time of the difference between lamellar body and surfactant specific activity against surfactant specific activity has a slope determined by the turnover time, and a shape which tests the precision of the precursor-product relationship. This analysis does not assume a pulse label and allows for possible recycling of tracer from surfactant to lamellar bodies. We obtained turnover times of 4–11 h. We detected an imprecise precursor-product relationship between lamellar bodies and alveolar surfactant, which is not due to experimental variability or to contamination of lamellar bodies by other subcellular fractions but may reflect imperfect mixing within surfactant compartments.

Because previous studies have suggested that lung surfactant is not a simple compartment of homogeneous material, we subfractionated lamellar bodies and components of alveolar lavage from male New Zealand white rabbits, according to differences in sedimentability. We recovered two lamellar body populations at different densities in discontinuous sucrose density gradients; we separated six subfractions of alveolar lavage by differential centrifugation. To determine whether or not precursor-product relationships existed among the subfractions, we injected radioactive palmitate intravenously, killed the rabbits 1–72 h later, and measured phospholipid specific activities. The two populations of lamellar bodies had similar phospholipid composition, fatty acyl composition of phosphatidylcholine and phosphatidylglycerol, and surface activity. Light lamellar bodies had a higher ratio of phospholipid to protein, and labelled with tracer later in time than dense ones. For alveolar lavage subtractions, later labelling with tracer, lower adsorption rate and lower total protein and phosphatidylglycerol content seemed to correlate with decreasing average density and particle size as well as with the disappearance of tubular myelin structure and appearance of predominantly vesicular structure. The subtractions appear to be in a metabolic sequence in which heavier, more dense material is a precursor to lighter, less dense material. The results suggest that subfractions of surfactant are extensively recycled.

To clarify the metabolic cycle of a low-molecular-weight protein of surfactant (SP-C), we obtained alveolar surfactant from 3 day old rabbits killed 24 h after the tracheal administration of 32 P or l -[ 35 S]methionine (donors). Aliquots of this naturally labelled surfactant were administered into trachea to 3-day-old rabbits (recipients) which were killed after 1 min or 3, 8 or 24 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine in fractions of lung lavage fluid and in lung homogenate. We found that alveolar SP-C is turned over faster than saturated phosphatidylcholine, that alveolar macrophages do participate in the removal of SP-C and that SP-C does not enter the fraction of alveolar surfactant that remains unsedimented after ultracentrifugation. Considering the whole lung, SP-C and saturated phosphatidylcholine are turned over at a comparable speed.

Altered lung function in hyperlipidemic patients has been reported by many authors. An alteration of surfactant synthesis has been suggested. Isolated lungs of rats rendered hyperlipidemic by suitable diets display an increased distensibility at maximal inflation and a higher degree of alveolar stability during deflation. These alterations are related to modifications of surfactant properties. Lung lavage fluid obtained from hyperlipidemic rats displays an increase in percent content of phosphatidylglycerol and a decrease of phosphatidylethanolamine. The percent content of phosphatidylglycerol correlates with the circulating levels of free fatty acids (FFA). It is suggested that FFA might affect the activity of enzymes operating in lung phospholipid synthesis. The reported increase of surfactant phosphatidylglycerol might explain the increment of alveolar stability observed in hyperlipidemic rats.

In the lower respiratory tract, alveolar cells are exposed to an oxidative challenge related to the exposure to both high levels of molecular oxygen and oxidants generated by activated phagocytes. The antioxidant defence system of alveolar cells has been thoroughly investigated, but some reports also suggest the presence of antioxidants in the layer of fluid lining the alveoli. In this report we present our studies on the antioxidant activities present in the bronchoalveolar lavage of adult rabbits. We studied total radical-trapping antioxidant capacity of surfactant and the activity of antiperoxidant enzymes. Although previous reports suggested the presence of radical scavengers, we did not find any antioxidant activity in purified surfactant. On the other hand the alveolar-lining fluid seems to contain superoxide dismutase, catalase and glutathione peroxidase, but not appreciable amounts of ferroxidase activity, as previously suggested. These enzymes could protect alveolar cells by catalyzing the dismutation of superoxide and hydrogen peroxide. The presence of glutathione peroxidase in the alveolar space seems to be physiologically relevant since the alveolar lining fluid also contains millimolar amounts of glutathione. Our studies support the concept that the alveolar lining fluid contains an active defence system against products of partial reduction of oxygen, but not chain-breaker antioxidants.

We report on clinical and metabolic studies of a newly delineated lipomatosis, characterised by an abnormal mediastinal and abdominal accumulation of fat, without obesity. The clinical features, which occurred in all the patients studied, are: Exertional dyspnoea due to a space-occupying mediastinal accumulation of fat, without evidence of cardiac or pulmonary disease. A pseudo-ascitic abdominal enlargement, due to intra- and retroperitoneal accumulation of fatty tissue. Insulin-independent diabetes mellitus. Type IV hyperlipidaemia and elevated levels of plasma uric acid were observed in four patients. Intra-abdominal lipomatous tissue, obtained during laparoscopy from four patients, demonstrated a reduced lipolytic response to beta-adrenergic stimulation. Thus, fat deposition in the abdominal and mediastinal areas could be causally related to defective lipid mobilization in lipomatocytes. Lipoprotein lipase activity in abdominal adipose tissue were normal in two patients (10.0 and 10.6 nmol/g/min) and markedly elevated in another two patients (37.3 and 49.9 nmol/g/min), as compared with controls (12.7 +/- 2.1 nmol/g/min). When expressed on per cell basis, LPL activity in lipomatous tissue was significantly higher than in control tissue (3.21 +/- 1.1 nmol/10(5) cell/min vs 0.92 +/- 0.16 nmol/10(5) cell/min). Lipoprotein fractionation did not demonstrate consistent modification of the serum lipoprotein pattern. HDL and HDL2 cholesterol values were reduced, even in patients with elevated LPL activity in adipose tissue.

Alveolar macrophages degrade surfactant protein (SP) A and saturated phosphatidycholine [dipalmitoylphosphatidylcholine (DPPC)]. To clarify this process, using rabbit alveolar macrophages, we analyzed the effect of drugs known to affect phagocytosis, pinocytosis, clathrin-mediated uptake, caveolae, the cytoskeleton, lysosomal pH, protein kinase C, and phosphatidylinositol 3-kinase (PI3K) on the degradation of SP-A and DPPC. We found the following: 1) SP-A binds to the plasma membrane, is rapidly internalized, and then moves toward degradative compartments. Uptake could be clathrin mediated, whereas phagocytosis, pinocytosis, or the use of caveolae are less likely. An intact cytoskeleton and an acidic milieu are necessary for the degradation of SP-A. 2) Stimulation of protein kinase C increases the degradation of SP-A. 3) PI3K influences the degradation of SP-A by regulating both the speed of internalization and subsequent intracellular steps, but its inhibition does not prevent SP-A from reaching the lysosomal compartment. 4) The degradation of DPPC is unaffected by most of the treatments able to influence the degradation of SP-A. Thus it appears that DPPC is degraded by alveolar macrophages through mechanisms very different from those utilized for the degradation of SP-A.

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.

To clarify the steps following surfactant secretion, we injected adult rabbits with radioactive palmitate, lavaged the airways, removed the cells, separated by ultracentrifugation lavage components into two fractions (B and C), and followed the labeling of phospholipids of these fractions. The results were compatible with the view that total and saturated phosphatidylcholine are transferred from B to C. Furthermore, the fluxes of total and saturated phosphatidylcholine through fraction C (0.45 and 0.30 mumol . h-1 . g lung-1, respectively) were compatible with the actual estimates of surfactant recycling. The labeling of phosphatidylglycerol ruled out a simple precursor-product relationship between B and C but was compatible with a nonideal first-order relationship. The labeling of phosphatidylinositol, cardiolipin, and phosphatidylethanolamine was incompatible with the existence of a direct precursor-product relationship between B and C. The labeling of total and saturated phosphatidylcholine suggests that fraction B may be made by active surfactant, whereas fraction C may contain surfactant modified for reuptake or for reuptake and catabolism.

To clarify the life cycle of surfactant protein C (SP-C), we obtained alveolar surfactant from 3-day-old rabbits killed 15 h after the tracheal administration of 32P and L-[35S]methionine. Small amounts of this surfactant were administered into the tracheas of 3-day-old rabbits, which were killed after 1 min and 2 and 4 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine (DPPC) in lung lavage fluid and in a lamellar body-rich fraction isolated from the lung homogenate. We found that SP-C and DPPC are both taken up from the alveolar spaces and incorporated into lamellar bodies, but the time courses of reuptake are different. Our results indicate that in the alveoli SP-C is turned over faster than DPPC.