Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures.

Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (lamellar body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process governing the size and hydration of these intracellular LB-like structures. Specifically, a first step (days 1 to 7) is characterized by an increase in their size, followed by a second one (days 7 to 14) where the organelles display a decrease in their global hydration properties. Interestingly, our results also show that their hydration properties significantly differ from those observed in well-characterized artificial lamellar model membranes, challenging the notion that a pure lamellar membrane organization is present in these organelles at intracellular conditions. Finally, these LB-like structures show a significant increase in their hydration state upon secretion, suggesting a relevant role of entropy during this process.

Organizational Issues, Structure, and Processes of Care in 257 ICUs in Latin America: A Study From the Latin America Intensive Care Network.

OBJECTIVE: Latin America bears an important burden of critical care disease, yet the information about it is scarce. Our objective was to describe structure, organization, processes of care, and research activities in Latin-American ICUs. DESIGN: Web-based survey submitted to ICU directors. SETTINGS: ICUs located in nine Latin-American countries. SUBJECTS: Individual ICUs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Two hundred fifty-seven of 498 (52%) of submitted surveys responded: 51% from Brazil, 17% Chile, 13% Argentina, 6% Ecuador, 5% Uruguay, 3% Colombia, and 5% between Mexico, Peru, and Paraguay. Seventy-nine percent of participating hospitals had less than 500 beds; most were public (59%) and academic (66%). ICUs were mainly medical-surgical (75%); number of beds was evenly distributed in the entire cohort; 77% had 24/7 intensivists; 46% had a physician-to-patient ratio between 1:4 and 7; and 69% had a nurse-to-patient ratio of 1 ≥ 2.1. The 24/7 presence of other specialists was deficient. Protocols in use averaged 9 ± 3. Brazil (vs the rest) had larger hospitals and ICUs and more quality, surveillance, and prevention committees, but fewer 24/7 intensivists and poorer nurse-to-patient ratio. Although standard monitoring, laboratory, and imaging practices were almost universal, more complex measurements and treatments and portable equipment were scarce after standard working hours, and in public hospitals. Mortality was 17.8%, without differences between countries. CONCLUSIONS: This multinational study shows major concerns in the delivery of critical care across Latin America, particularly in human resources. Technology was suboptimal, especially in public hospitals. A 24/7 availability of supporting specialists and of key procedures was inadequate. Mortality was high in comparison to high-income countries.

Sevoflurane anesthesia deteriorates pulmonary surfactant promoting alveolar collapse in male Sprague-Dawley rats.

General anesthesia is frequently associated to transient hypoxemia and lung atelectasis. Although volatile anesthetics are safe and widely used, their potential role on anesthesia-induced pulmonary impairment has not been fully explored. In this study, we investigated the effect of volatile anesthetic sevoflurane on pulmonary surfactant composition and structure that could contribute to atelectasis. After 30 min of sevoflurane anesthesia, Sprague-Dawley rats showed increased levels of lyso-phosphatidylcholine and decreased levels of phosphatidylcholine associated with significant impairment in lung mechanics and alveolar collapse, but showed no deterioration of alveolar fluid reabsorption when compared to control group of rats anesthetized with pentobarbital. Exposure to sevoflurane altered the thermotropic profile of surfactant model membranes, as detected by fluorescence anisotropy. In this sense, sevoflurane-promoted fluidification of condensed phases could potentially impair the ability of surfactant films to sustain the lowest surface tensions. In conclusion, the observed changes in surfactant composition and viscosity properties suggest a direct effect of sevoflurane on surfactant function, a factor potentially involved in anesthetic-induced alterations in lung mechanics.

Picking up the pieces: towards a better future for critical care medicine in three South American countries.

The demand for intensivists is increasing around the world, not only to meet the needs of a growing aging population, but also to fill positions in the intensive care unit now occupied by other specialists, since there is compelling evidence that the presence of critical care practitioners improves patient outcomes. Notwithstanding this, the shortage of intensivists is a problem recognized throughout the world. In this article, we discuss these issues in Argentina, Brazil, and Uruguay, three upper-middle income Latin American countries where critical care has been a medical specialty for decades and intensive care unit coverage traditionally has followed the 24/7 model. The lack of intensivists is multifactorial: the specialty is not taught to medical students; there is a general perception of a negative lifestyle compared with the practice of other medical specialties, due mainly to the constant 24-hour shift work; and there is general dissatisfaction with incomes, which has forced many intensivists into multijob schemes. The expected-and feared-consequences are the 40 to 70% vacant posts in residencies of critical care. Despite these drawbacks, scientific societies and colleges are intensely committed, pointing out these problems to the press, calling the health authorities for action, and permanently generating educational activities. Surprisingly, 83% of surveyed intensivists would choose critical care medicine again, evidencing the strong vocational component in its practice, which seems to predominate over negative aspects.

Novel opportunities from bioimaging to understand the trafficking and maturation of intracellular pulmonary surfactant and its role in lung diseases.

Pulmonary surfactant (PS), a complex mixture of lipids and proteins, is essential for maintaining proper lung function. It reduces surface tension in the alveoli, preventing collapse during expiration and facilitating re-expansion during inspiration. Additionally, PS has crucial roles in the respiratory system's innate defense and immune regulation. Dysfunction of PS contributes to various respiratory diseases, including neonatal respiratory distress syndrome (NRDS), adult respiratory distress syndrome (ARDS), COVID-19-associated ARDS, and ventilator-induced lung injury (VILI), among others. Furthermore, PS alterations play a significant role in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The intracellular stage involves storing and releasing a specialized subcellular organelle known as lamellar bodies (LB). The maturation of these organelles requires coordinated signaling to organize their intracellular organization in time and space. LB's intracellular maturation involves the lipid composition and critical processing of surfactant proteins to achieve proper functionality. Over a decade ago, the supramolecular organization of lamellar bodies was studied using electron microscopy. In recent years, novel bioimaging tools combining spectroscopy and microscopy have been utilized to investigate the in cellulo intracellular organization of lamellar bodies temporally and spatially. This short review provides an up-to-date understanding of intracellular LBs. Hyperspectral imaging and phasor analysis have allowed identifying specific transitions in LB's hydration, providing insights into their membrane dynamics and structure. A discussion and overview of the latest approaches that have contributed to a new comprehension of the trafficking and structure of lamellar bodies is presented.

Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt.

Stimulation of Na(+)/K(+)-ATPase translocation to the cell surface increases active Na(+) transport, which is the driving force of alveolar fluid reabsorption, a process necessary to keep the lungs free of edema and to allow normal gas exchange. Here, we provide evidence that insulin increases alveolar fluid reabsorption and Na(+)/K(+)-ATPase activity by increasing its translocation to the plasma membrane in alveolar epithelial cells. Insulin-induced Akt activation is necessary and sufficient to promote Na(+)/K(+)-ATPase translocation to the plasma membrane. Phosphorylation of AS160 by Akt is also required in this process, whereas inactivation of the Rab GTPase-activating protein domain of AS160 promotes partial Na(+)/K(+)-ATPase translocation in the absence of insulin. We found that Rab10 functions as a downstream target of AS160 in insulin-induced Na(+)/K(+)-ATPase translocation. Collectively, these results suggest that Akt plays a major role in Na(+)/K(+)-ATPase intracellular translocation and thus in alveolar fluid reabsorption.

Peripheral and respiratory muscle impairment during murine acute lung injury.

Acute respiratory distress syndrome is associated with skeletal muscle compromise, which decreases survival and impairs functional capacity. A comparative analysis of peripheral and respiratory muscles' atrophy and dysfunction in acute lung injury (ALI) has not been performed. We aimed to evaluate diaphragmatic and peripheral muscle mass and contractility in an ALI animal model. ALI was induced in C57BL/6 mice by intratracheal lipopolysaccharides instillation. Muscle mass and in vitro contractility were evaluated at different time points in hindlimb soleus (slow-twitch) and extensor digitorum longus (EDL, fast-twitch), as well as in the main respiratory muscle diaphragm. Myogenic precursor satellite cell-specific transcription factor Pax7 expression was determined by Western blot. Lung injury was associated with atrophy of the three studied muscles, although it was more pronounced and persistent in the diaphragm. Specific contractility was reduced during lung injury in EDL muscle but restored by the time lung injury has resolved. Specific force was not affected in soleus and diaphragm. A persistent increase in Pax7 expression was detected in diaphragm and EDL muscles after induction of ALI, but not in soleus muscle. Different peripheral and respiratory skeletal muscles are distinctly affected during the course of ALI. Each of the studied muscles presented a unique pattern in terms of atrophy development, contractile dysfunction and Pax7 expression.

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis.

Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.

Adenosine triphosphate-dependent calcium signaling during ventilator-induced lung injury is amplified by hypercapnia.

Adenosine triphosphate (ATP) is released by alveolar epithelial cells during ventilator-induced lung injury (VILI) and regulates fluid transport across epithelia. High CO(2) levels are observed in patients with "permissive hypercapnia," which inhibits alveolar fluid reabsorption (AFR) in alveolar epithelial cells. The authors set out to determine whether VILI affects AFR and whether the purinergic pathway is modulated in cells exposed to hypercapnia. Control group was compared against VILI (tidal volume [Vt] = 35 mL/kg, zero positive end-expiratory pressure [PEEP]) and protective ventilation (Vt = 6 mL/kg, PEEP = 10 cm H(2)O) groups. Lung mechanics, histology, and AFR were evaluated. Alveolar epithelial cells (AECs) were loaded with Fura 2-AM to measure intracellular calcium in the presence ATP (10 µM) at 5% or 10% CO(2) as compared with baseline. High tidal volume ventilation impairs lung mechanics and AFR. Hypercapnia (HC) increases intracellular calcium levels in response to ATP stimulation. HC + ATP is the most detrimental combination decreasing AFR. Purinergic signaling in AECs is modulated by high CO(2) levels via increased cytosolic calcium. The authors reason that this modulation may play a role in the impairment of alveolar epithelial functions induced by hypercapnia.

Endothelin-1 impairs alveolar epithelial function via endothelial ETB receptor.

RATIONALE: Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR). OBJECTIVES: To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation. METHODS: Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake. MEASUREMENTS AND MAIN RESULTS: The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor. CONCLUSIONS: We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.

Fatal acute pancreatitis complicated by pancreatic pseudocysts in a patient with systemic lupus erythematosus.

Pancreatitis is a relatively rare but severe manifestation in systemic lupus erythematosus (SLE) patients. We report a case of a 39-year-old woman with previous SLE diagnose treated with prednisone and mycophenolate mofetil who developed an acute pancreatitis complicated by pancreatic pseudocysts within the context of a severe lupus flare. Elevated serum amylase and computerized tomography confirmed the diagnosis and mechanical obstruction or toxic-metabolic etiologies were ruled out. In the present case, we opted for the clinical surveillance of pancreatic pseudocyst and not perform invasive medical procedures to drainage. A steroid therapy was started in order to achieve SLE and pancreatitis remission, however, it was unable to avoid the development of multiorgan failure and patient died a few days after diagnosis was made.

Carbonic anhydrase II and alveolar fluid reabsorption during hypercapnia.

Carbonic anhydrase II (CAII) plays an important role in carbon dioxide metabolism and intracellular pH regulation. In this study, we provide evidence that CAII is expressed in both type I (AECI) and type II (AECII) alveolar epithelial cells by RT-PCR and Western blotting in freshly isolated rat cells. These results were further confirmed by double immunostaining with CAII antibodies and AECI- or AECII-specific markers in freshly isolated alveolar epithelial cells and rat lung tissues. Inhibition of CAII by acetazolamide or methazolamide delayed the decrease in the intracellular pH observed during hypercapnia in cultured AECI, AECII, and AECI-like cells. In an isolated-perfused rat lung model, alveolar fluid reabsorption significantly decreased during high CO(2) exposure, which was not prevented by carbonic anhydrase inhibition. Thus, we provide evidence that CAII is expressed in rat alveolar epithelial cells and does not regulate lung alveolar fluid reabsorption during hypercapnia.

Effects of hypercapnia in acute respiratory distress syndrome.

In patients with acute respiratory distress syndrome (ARDS) hypercapnia is a marker of poor prognosis, however there is controversial information regarding the effect of hypercapnia on outcomes. Recently two studies in a large population of mechanical ventilation patients showed higher mortality associated independently to hypercapnia. Key roles responsible for the poor clinical outcomes observed in critically ill patients exposed to hypercapnia are not well known, two possible mechanisms involved are the effect of CO2 on the muscle and the alveolar epithelium. Hypercapnia frequently coexists with muscle atrophy and dysfunction, moreover patients surviving ARDS present reduced muscle strength and decreased physical quality of life. One of the possible mechanisms responsible for these abnormalities could be the effects of hypercapnia during the course of ARDS. More over controversy persists about the hypercapnia role in the alveolar space, in the last years there is abundant experimental information on its deleterious effects on essential functions of the alveolar epithelium.

Lung protection: an intervention for tidal volume reduction in a teaching intensive care unit. / Proteção pulmonar: intervenção para reduzir o volume corrente em uma unidade de terapia intensiva de ensino.

OBJECTIVE:: To determine the effect of feedback and education regarding the use of predicted body weight to adjust tidal volume in a lung-protective mechanical ventilation strategy. METHODS:: The study was performed from October 2014 to November 2015 (12 months) in a single university polyvalent intensive care unit. We developed a combined intervention (education and feedback), placing particular attention on the importance of adjusting tidal volumes to predicted body weight bedside. In parallel, predicted body weight was estimated from knee height and included in clinical charts. RESULTS:: One hundred fifty-nine patients were included. Predicted body weight assessed by knee height instead of visual evaluation revealed that the delivered tidal volume was significantly higher than predicted. After the inclusion of predicted body weight, we observed a sustained reduction in delivered tidal volume from a mean (standard error) of 8.97 ± 0.32 to 7.49 ± 0.19mL/kg (p < 0.002). Furthermore, the protocol adherence was subsequently sustained for 12 months (delivered tidal volume 7.49 ± 0.54 versus 7.62 ± 0.20mL/kg; p = 0.103). CONCLUSION:: The lack of a reliable method to estimate the predicted body weight is a significant impairment for the application of a worldwide standard of care during mechanical ventilation. A combined intervention based on education and repeated feedbacks promoted sustained tidal volume education during the study period (12 months).

Prueba de ventilación espontánea en pacientes ventilados: evaluación del cumplimiento de pautas protocolizadas contra análisis del equipo asistencial / Spontaneous ventilation test in ventilated patients: an assessment of fulfillment of protocol guidelines vs the analysis by the health team

Introducción: la duración ideal de la asistencia ventilatoria mecánica (AVM) genera debate e incertidumbre. El intento de desvinculación prematuro condiciona fracaso y aumento de mortalidad, mientras que la desvinculación tardía aumenta el riesgo del paciente. La utilización de pruebas de ventilación espontánea (PVE) son seguras e identifican aceptablemente a los pacientes listos para desvinculación. Sin embargo, la adherencia a pautas protocolizadas es muy variable y falla la implementación de evidencia científica a nivel clínico. Objetivo: analizar la interacción entre pautas de desvinculación de AVM y evaluación médica para la toma de decisiones. Material y método: trabajo descriptivo, prospectivo, con entrevista a médicos en la unidad de cuidados intensivos (UCI) sobre su valoración de la condición del paciente para realizar una PVE. Se comparó la opinión del médico con la evaluación hecha según protocolo de la UCI. Se analizaron coincidencias y discrepancias entre opinión de médicos y protocolo. Resultados: ingresaron 27 pacientes y 46 médicos. Las coincidencias representaron el 85,4% de las opiniones aunque existieron elementos de "confusión" en la decisión médica, tanto en coincidencias como en discrepancias. El más frecuente fue el estado de conciencia del paciente. Discusión y conclusiones: la valoración de la conciencia es fundamental para la asistencia diaria, pero no para la PVE. Su inclusión se vio involucrada en casi un tercio de las respuestas obtenidas, difiriendo la realización de la PVE. Este aspecto debe ser tenido en cuenta tanto para instancias docentes como asistenciales al momento de optimizar los tiempos para PVE y desvinculación de la AVM.

Introduction: Ideal duration of mechanical ventilation is a source of debate and uncertainty. Early weaning attempts result in failure and increased mortality rates, while a late discontinuation of ventilation increases the patients' risks. The use of the spontaneous ventilation test is safe and results in a fair identification of patients who are ready for weaning. However, adherence to protocol guidelines varies and scientific evidence fails to be implemented in the clinical practice. Objective: to analyze the interaction between mechanical ventilation discontinuation guidelines and medical assessment for the making of a decision. Method: descriptive, prospective study which involved interviewing physicians in the Intensive Care Unit on their assessment of the patient's condition to perform a spontaneous ventilation test. The physicians´ opinion was compared to the assessment carried out as per the Intensive Care Unit Protocol, in order to analyze agreements and discrepancies. Results: 27 patients and 46 physicians were included in the study. Agreements represented 85.4% of opinions, although there were a few confusing elements confusing as to the medical decision to be made, both in terms of agreements and discrepancies, the most frequent of which was the patient's level of consciousness. Discussion and conclusions: assessing the level of consciousness of patients is essential in the daily practice of medicine, although not for the spontaneous ventilation test. It was included in almost one third of the responses obtained and delayed the performance of a spontaneous ventilation test. This fact needs to be considered both in the context of training instances and at the time of optimizing times for the spontaneous ventilation test and the discontinuation of mechanical ventilation.

Introdução: a duração ideal da Assistência Ventilatória Mecânica (AVM) gera debate e incerteza. A tentativa de desmame precoce leva ao fracasso e ao aumento da mortalidade, enquanto o desmame tardio aumenta o risco do paciente. A utilização de testes de ventilação espontânea (TVE) são seguras e identificam razoavelmente os pacientes que já estão em condições para o desmame. No entanto, a adesão a pautas protocolizadas é muito variável e a implementação de evidencia científica no nível clínico falha. Objetivo: analisar a interação entre as pautas de desmame de AVM e a avaliação médica para a tomada de decisões. Material e método: trabalho descritivo, prospectivo, com entrevista a médicos na Unidade de Cuidados Intensivos (UCI) sobre sua avaliação da condição do paciente para realizar um TVE. A opinião do médico e a avaliação feita de acordo com o protocolo da UCI foram comparadas. As coincidências e discrepâncias entre opinião de médicos e protocolo foram analisadas. Resultados: 27 pacientes e 46 médicos foram incluídos. As coincidências representaram 85,4%, das opiniões mesmo quando se observaram elementos de "confusão" na decisão médica, tanto em coincidências como discrepâncias. O mais frequente estava relacionado com o estado de consciência do paciente. Discussão e conclusões: a avaliação da consciência é fundamental para a assistência diária, mas não para o TVE. Sua inclusão estava presente em quase um terço das respostas obtidas, com diferencias sobre a realização do TVE. Este aspecto deve ser considerado tanto na docência como na assistência para otimizar os tempos para a realização de TVE e do desmame da AVM.

Evolutionary conserved role of c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction.

Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes.