Molecular and biophysical mechanisms behind the enhancement of lung surfactant function during controlled therapeutic hypothermia.

Therapeutic hypothermia (TH) enhances pulmonary surfactant performance in vivo by molecular mechanisms still unknown. Here, the interfacial structure and the composition of lung surfactant films have been analysed in vitro under TH as well as the molecular basis of its improved performance both under physiological and inhibitory conditions. The biophysical activity of a purified porcine surfactant was tested under slow and breathing-like dynamics by constrained drop surfactometry (CDS) and in the captive bubble surfactometer (CBS) at both 33 and 37 °C. Additionally, the temperature-dependent surfactant activity was also analysed upon inhibition by plasma and subsequent restoration by further surfactant supplementation. Interfacial performance was correlated with lateral structure and lipid composition of films made of native surfactant. Lipid/protein mixtures designed as models to mimic different surfactant contexts were also studied. The capability of surfactant to drastically reduce surface tension was enhanced at 33 °C. Larger DPPC-enriched domains and lower percentages of less active lipids were detected in surfactant films exposed to TH-like conditions. Surfactant resistance to plasma inhibition was boosted and restoration therapies were more effective at 33 °C. This may explain the improved respiratory outcomes observed in cooled patients with acute respiratory distress syndrome and opens new opportunities in the treatment of acute lung injury.

Supramolecular Assembly of Human Pulmonary Surfactant Protein SP-D.

Pulmonary surfactant protein D (SP-D) is a glycoprotein from the collectin family that is a component of the lung surfactant system. It exhibits host defense and immune regulatory functions in addition to contributing to the homeostasis of the surfactant pool within the alveolar airspaces. It is known that the SP-D monomer forms trimers, which further associate into higher-order oligomers. However, the pathway and the interactions involved in the assembly of SP-D oligomers are not clearly understood. In the current study, a recombinant form of full-length human SP-D (rhSP-D) has been qualitatively and quantitatively studied by atomic force microscopy (AFM) and electrophoresis, with the aim to understand the conformational diversity and the determinants defining the oligomerization of the protein. The rhSP-D preparation studied is a mixture of trimers, hexamers, dodecamers and higher-order oligomeric species, with dodecamers accounting for more than 50% of the protein by mass. Similar structures were also found in hSP-D obtained from proteinosis patients, with the largest fuzzy-ball-like oligomers being more abundant in these samples. The proportion of dodecamer is increased under acidic conditions, accompanied by a conformational change into more compact configurations. Two hexamers appear to be the minimal necessary unit for dodecamer formation, with stabilization of the dodecamer occurring via non-covalent, ionic, and hydrophobic interactions between the individual N-terminal domains and the proximal area of the SP-D collagen stems.

Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted ß-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form ß-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.

Neuroma en la incisión de Pfannestiel como causa de un dolor abdominal intratable / Traumatic neuroma under Pfannestiel’s incision as a cause of untreatable abdominal pain

El neuroma es un crecimiento disperso y desordenado de las fibras nerviosas entremezcladas en tejido fibroso durante su proceso de regeneración. Es una patología poco común en la práctica clínica, y su diagnóstico en ocasiones resulta dificultoso y demorado en el tiempo. Presentamos el caso de una paciente de 13 años de edad con un cuadro de dolor abdominal incoercible y sin alteraciones en las pruebas diagnósticas realizadas, cuya causa resultó un neuroma en la cicatriz quirúrgica previa (AU)

The neuroma is a dispersed and disordered growth of the nervous fibers intermingled with fibrous tissue during its process of regeneration. It is a fairly uncommon pathology in the clinical practice and the diagnosis sometimes proves to be difficult and delayed in time. The author presents the clinical case of a 13-year-old patient with a severe abdominal pain, all diagnostic tests being normal without any alterations. The cause of the abdominal pain was a neuroma in a previous surgical scar (AU)

New surfactant with SP-B and C analogs gives survival benefit after inactivation in preterm lambs.

BACKGROUND: Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if P(a)O(2) dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals. CONCLUSIONS: For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.

Phase-field model for the morphology of monolayer lipid domains.

Phase-separated domains exist in multicomponent lipid monolayers and bilayers. We present here a phase-field model that takes into account the competition between lipid dipole-dipole interactions and line tension to define the domain morphology. A dynamic equation for the phase-field is solved numerically showing stationary non-circular shapes like starfish shapes. This phase-field model could be applied to study the dynamic properties of complex problems like phase segregation in pulmonary surfactant membranes and films.

Complicaciones tras esofagoscopia rígida y posterior colocación de sonda nasogástrica / No disponible

No disponible

[Use of a metal guide in the working channel of a fiberoptic scope to insert a tracheal tube in an infant with Treacher Collins syndrome and choanal atresia]. / Uso de guía metálica para intubación traqueal mediante fibroscopia en un neonato con síndrome de Treacher Collins y atresia de coanas.

Neonates with Treacher Collins syndrome can present difficult airways. Ventilation through a face mask and laryngoscopy for tracheal intubation may prove impossible due to the craniofacial malformations that are characteristic of this syndrome. Furthermore, patients with this syndrome are at high risk of airway obstruction, meaning that awake fiberoptic endoscopy provides the best option for tracheal intubation. This technique is especially difficult in children, however, and material required for performing it in neonates is not always available. We report the case of a 5-day-old infant boy with Treacher Collins syndrome and bilateral choanal atresia in whom we used a flexible metal guide inserted into the working channel of a fiberoptic scope. The tracheal tube could then be inserted.

Uso de guía metálica para intubación traqueal mediante fibroscopia en un neonato con síndrome de Treacher Collins y atresia de coanas / Use of a metal guide in the working channel of a fiberoptic scope to insert a tracheal tube in an infant with Treacher Collins syndrome and choanal atresia

Los neonatos afectos de síndrome de Treacher Collinspueden presentar dificultades en el manejo de la víaaérea. Las malformaciones a nivel del macizo craneofacial,características de este síndrome, implican que laventilación con mascarilla facial y la laringoscopia paraintubación orotraqueal puedan resultar imposibles. Además,el mayor riesgo de obstrucción de la vía aérea quepresentan estos pacientes hace que sea la fibroscopia,con el paciente despierto, el método más adecuado parala intubación orotraqueal. Esta técnica es especialmentedifícil en la población pediátrica y no siempre se disponede material específico para neonatos. Presentamos elcaso de un niño de 5 días de vida con síndrome de TreacherCollins y atresia coanal bilateral en el que se utilizóuna guía metálica flexible, introducida a través del canalde trabajo del fibroscopio para seguidamente insertar eltubo orotraqueal a través de dicha guía(AU)

Neonates with Treacher Collins syndrome can presentdifficult airways. Ventilation through a face mask andlaryngoscopy for tracheal intubation may proveimpossible due to the craniofacial malformations thatare characteristic of this syndrome. Furthermore,patients with this syndrome are at high risk of airwayobstruction, meaning that awake fiberoptic endoscopyprovides the best option for tracheal intubation. Thistechnique is especially difficult in children, however, andmaterial required for performing it in neonates is notalways available. We report the case of a 5-day-oldinfant boy with Treacher Collins syndrome and bilateralchoanal atresia in whom we used a flexible metal guideinserted into the working channel of a fiberoptic scope.The tracheal tube could then be inserted(AU)

[Scaling methods applied to set priorities in training programs in organizations]. / Métodos de escalamiento aplicados a la priorización de necesidades de formación en organizaciones.

Criteria to assess the needs in order to plan training programs are not usually defined explicitly in organizational contexts. We propose scaling methods as a feasible and useful procedure to set priorities of training programs; also, we propose the most suitable method for this intervention context. 404 employees from a public organization completed an ad hoc questionnaire to assess training needs in different areas from 2004 to 2006; concretely, 117, 75 and 286 stimuli were scaled, respectively. Then, four scaling methods were compared: Dunn-Rankin's method and three methods derived from Thurstone's Law of Categorical Judgment--ranking, successive intervals and equal-appearing intervals. The feasibility and utility of these scaling methods to solve the problems described is shown. Taking into account the most accurate compared methods, we propose ranking as the most parsimonious method (with regard to procedure simplicity). Future research developments are described.

Surfactant protein SP-B strongly modifies surface collapse of phospholipid vesicles: insights from a quartz crystal microbalance with dissipation.

Pulmonary surfactant protein B (SP-B) facilitates the rapid transfer of phospholipids from bilayer stores into air-liquid interfacial films along the breathing cycle, and contributes to the formation of a surface-associated multilayer reservoir of surfactant to optimize the stability of the respiratory interface. To obtain more insights into the mechanisms underlying this transfer and multilayer formation, we established a simple model system that captures different features of SP-B action. We monitored the formation of supported planar bilayers from the collapse of intact phospholipid vesicles on a silica surface using a technique called quartz crystal microbalance with dissipation, which provides information on changes in membrane thickness and viscosity. At physiologically relevant concentrations, SP-B dramatically alters vesicle collapse. This manifests itself as a reduced buildup of intact vesicles on the surface before collapse, and allows the stepwise buildup of multilayered deposits. Accumulation of lipids in these multilayer deposits requires the presence of SP-B in both the receptor and the arriving membranes, surrounded by a comparable phospholipid charge. Thus, the quartz crystal microbalance with dissipation system provides a useful, simplified way to mimic the effect of surfactant protein on vesicle dynamics and permits a detailed characterization of the parameters governing reorganization of surfactant layers.

Cholesterol modulates the exposure and orientation of pulmonary surfactant protein SP-C in model surfactant membranes.

Cholesterol is the major neutral lipid in lung surfactant, accounting for up to 8-10% of surfactant mass, while surfactant protein SP-C ( approximately 4.2 kDa) accounts for no more than 1-1.5% of total surfactant weight but plays critical roles in formation and stabilization of pulmonary surfactant films. It has been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films and this interaction could have a potential role on modulating surfactant function. In the present study, we have analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes. The presence of cholesterol does not induce substantial changes in the secondary structure of SP-C, as analyzed by Attenuated Reflection Fourier Transformed Infrared spectroscopy (ATR-FTIR). However, the presence of cholesterol modifies the orientation of the transmembrane helix and the dynamic properties of the protein, as demonstrated by hydrogen/deuterium exchange kinetics. The effect of cholesterol on SP-C reconstituted in zwitterionic, entirely fluid, membranes made of POPC (palmitoyloleoylphospatidylcholine) or in anionic membranes with coexistence of ordered and disordered phases, such as those made of dipalmitoylphosphatidylcholine (DPPC):POPC:Palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15) is dual. Cholesterol decreases the exposure of the protein to the aqueous environment and the tilt of its transmembrane helical segment up to a ratio Cholesterol:SP-C of 4.8 and 2.4 (mol/mol) in the two lipid systems tested, respectively, and it increases the exposure and tilt at higher cholesterol proportions. The results presented here suggest the existence of an interaction between SP-C and cholesterol-enriched phases, with consequences on the behavior of the protein, which could be of relevance for cholesterol-dependent structure-function relationships in pulmonary surfactant membranes and films.

Métodos de escalamiento aplicados a la priorización de necesidades de formación en organizaciones / Scaling methods applied to set priorities in training programs in organizations

Los criterios para priorizar las necesidades que justifican las acciones formativas a implantar no se suelen explicitar a priori en los programas de formación continua en contextos organizacionales. En este trabajo se proponen los métodos de escalamiento como procedimiento factible y útil para identificar criterios explícitos de priorización de necesidades, y se concreta cuál de ellos es más apropiado en este contexto de intervención. 404 empleados de una organización pública cumplimentaron un cuestionario ad hoc para priorizar necesidades formativas en diferentes áreas durante el período 2004 al 2006; concretamente, se ordenaron 117, 75 y 286 estímulos, respectivamente. Se calcularon y compararon las ordenaciones obtenidas con cuatro métodos de escalamiento: el método de Dunn-Rankin y tres métodos derivados de la Ley del Juicio Categórico de Thurstone, concretamente ordenación por rangos, intervalos sucesivos e intervalos aparentemente iguales. Con los resultados, se constata la factibilidad y utilidad de estos métodos de escalamiento para la solución de los problemas planteados; a partir delos métodos más precisos, se propone el uso del método de ordenación por rangos por su parsimonia (respecto a sencillez en su procedimiento); y se anticipan futuras líneas de actuación (AU)

Criteria to assess the needs in order to plan training programs are not usually defined explicitly in organizational contexts.We propose scaling methods as a feasible and useful procedure to set priorities of training programs; also, we propose the most suitable method for this intervention context. 404 employees from a public organization completed an ad hoc questionnaire to assess training needs in different areas from 2004 to 2006; concretely, 117, 75 and 286 stimuli were scaled, respectively. Then, four scaling methods were compared: Dunn-Rankin’s method and three methods derived from Thurstone’s Law of Categorical Judgment -ranking, successive intervals and equal-appearing intervals. The feasibility and utility of these scaling methods to solve the problems described is shown. Taking into account the most accurate compared methods, we propose ranking as the most parsimonious method (with regard to procedures implicity). Future research developments are described (AU)

Self-aggregation of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study.

A recombinant form of the peptide N-terminally positioned from proSP-B (SP-BN) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and purified thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidified secretory pathway of pneumocytes. Self-aggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and finally basic. The profile of aggregation versus subsequent changes in pH showed differences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-BN samples along the aggregation process showed a decrease in alpha-helical content and a concomitant increase in beta-sheet. Intrinsic fluorescence emission of SP-BN was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the fluorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-BN at pH 5. The fluorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-BN module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes.

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane alpha-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.

Interfacial properties of pulmonary surfactant layers.

The composition of the pulmonary surfactant and the border conditions of normal human breathing are relevant to characterize the interfacial behavior of pulmonary layers. Based on experimental data methods are reviewed to investigate interfacial properties of artificial pulmonary layers and to explain the behavior and interfacial structures of the main components during compression and expansion of the layers observed by epifluorescence and scanning force microscopy. Terms like over-compression, collapse, and formation of the surfactant reservoir are discussed. Consequences for the viscoelastic surface rheological behavior of such layers are elucidated by surface pressure relaxation and harmonic oscillation experiments. Based on a generalized Volmer isotherm the interfacial phase transition is discussed for the hydrophobic surfactant proteins, SP-B and SP-C, as well as for the mixtures of dipalmitoylphosphatidylcholine (DPPC) with these proteins. The behavior of the layers depends on both the oligomerisation state and the secondary structure of the hydrophobic surfactant proteins, which are controlled by the preparation of the proteins. An example for the surface properties of bronchoalveolar porcine lung washings of uninjured, injured, and Curosurf treated lavage is discussed in the light of surface behavior. An outlook summarizes the present knowledge and the main future development in this field of surface science.

Potassium efflux induced by a new lactoferrin-derived peptide mimicking the effect of native human lactoferrin on the bacterial cytoplasmic membrane.

A 31-amino acid synthetic peptide (NH(2)-FFSASCVPGADKGQFPNLCRLCAGTGENKCA-COOH) was chemically synthesized based on the amino acid sequence of a region of human lactoferrin homologous to other sequences present in the N- and C-lobes of all members of the transferrin family proteins. The peptide, termed kaliocin-1, and lactoferrin showed a bactericidal effect in assays performed in low-ionic-strength conditions. This is the first time that it is shown that the antimicrobial effect of lactoferrin depends on the extracellular cation concentration. The antimicrobial effect of kaliocin-1 was lower than that of human lactoferrin, but their activities were inhibited by Na(+) or K(+) in a cation concentration-dependent manner. In addition, the peptide was able to mimic native lactoferrin, inducing K(+)-efflux and a selective dissipation of the transmembrane electrical potential of Escherichia coli cells without causing extensive damage to the outer and inner bacterial membranes. In contrast, the peptide, but not lactoferrin, was able to permeabilize different ions through liposomal membranes. The hypothetical interaction of kaliocin-1 with a bacterial membrane compound is discussed based in the different ion flux induced on cellular and artificial membranes as well as data from circular dichroism assays. Kaliocin-1 was not cytotoxic and could be a suitable model for the design of analogs able to mimic the antibacterial effect of human lactoferrin.

Effects of oligomerization and secondary structure on the surface behavior of pulmonary surfactant proteins SP-B and SP-C.

The relationship among protein oligomerization, secondary structure at the interface, and the interfacial behavior was investigated for spread layers of native pulmonary surfactant associated proteins B and C. SP-B and SP-C were isolated either from butanol or chloroform/methanol lipid extracts that were obtained from sheep lung washings. The proteins were separated from other components by gel exclusion chromatography or by high performance liquid chromatography. SDS gel electrophoresis data indicate that the SP-B samples obtained using different solvents showed different oligomerization states of the protein. The CD and FTIR spectra of SP-B isolated from all extracts were consistent with a secondary structure dominated by alpha-helix. The CD and FTIR spectra of the first SP-C corresponded to an alpha-helical secondary structure and the spectra of the second SP-C corresponded to a mixture of alpha-helical and beta-sheet conformation. In contrast, the spectra of the third SP-C corresponded to antiparallel beta-sheets. The interfacial behavior was characterized by surface pressure/area (pi-A) isotherms. Differences in the oligomerization state of SP-B as well as in the secondary structure of SP-C all produce significant differences in the surface pressure/area isotherms. The molecular cross sections determined from the pi-A isotherms and from dynamic cycling experiments were 6 nm(2)/dimer molecule for SP-B and 1.15 nm(2)/molecule for SP-C in alpha-helical conformation and 1.05 nm(2)/molecule for SP-C in beta-sheet conformation. Both the oligomer ratio of SP-B and the secondary structure of SP-C strongly influence organization and behavior of these proteins in monolayer assemblies. In addition, alpha-helix --> beta-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.