Methodological Aspects of Induced Sputum.

We aimed to conduct a state-of-the-art review of the current literature and offer further insights into the methodological aspects concerning induced sputum. The increasing popularity of sputum induction as a non-invasive and cost-effective method for obtaining lower airway secretions from patients who cannot produce sputum naturally has led to extensive research and applications in respiratory conditions like asthma and COPD. This technique allows for analysis of the cellular and biochemical components of the sputum to take place, providing insights into airway inflammation, immune cells, and help in predicting treatment response. Furthermore, induced sputum enables various analyses, including microRNA and gene expression studies and immunophenotyping. The procedure is generally safe and well tolerated, even in patients with airflow limitations; however, monitoring lung function is essential, especially in those with airway hyperresponsiveness. Optimal saline solution concentration and inhalation duration have been investigated, recommending a 15-20 min induction with hypertonic saline. Expectoration involves coughing at the end of each inhalation time. Careful handling during sputum processing is necessary for obtaining accurate results in cell cytology, immunocytochemistry, and in situ hybridization. Overall, induced sputum offers significant advantages as a preferred alternative for large-scale and repeated airway sampling, despite some technical demands and limitations.

Sodium regulates clock time and output via an excitatory GABAergic pathway.

The suprachiasmatic nucleus (SCN) serves as the body's master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day-night cycle1-4. For example, the SCN opposes overnight adipsia by driving water intake before sleep5,6, and by driving the secretion of anti-diuretic hormone7,8 and lowering body temperature9,10 to reduce water loss during sleep11. These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase12-16. However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19-a time at which SCNVP (vasopressin) neurons are inactive-excited SCNVP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCNVP neurons and mimicked by optogenetic stimulation of SCNVP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLTGAD) relay this information to SCNVP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLTGAD neuron axon terminals excited SCNVP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLTGAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLTGAD→ SCNVP pathway and was prevented by chemogenetic inhibition of OVLTGAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks.

The Medical Management of Cerebral Edema: Past, Present, and Future Therapies.

Cerebral edema is commonly associated with cerebral pathology, and the clinical manifestation is largely related to the underlying lesioned tissue. Brain edema usually amplifies the dysfunction of the lesioned tissue and the burden of cerebral edema correlates with increased morbidity and mortality across diseases. Our modern-day approach to the medical management of cerebral edema has largely revolved around, an increasingly artificial distinction between cytotoxic and vasogenic cerebral edema. These nontargeted interventions such as hyperosmolar agents and sedation have been the mainstay in clinical practice and offer noneloquent solutions to a dire problem. Our current understanding of the underlying molecular mechanisms driving cerebral edema is becoming much more advanced, with differences being identified across diseases and populations. As our understanding of the underlying molecular mechanisms in neuronal injury continues to expand, so too is the list of targeted therapies in the pipeline. Here we present a brief review of the molecular mechanisms driving cerebral edema and a current overview of our understanding of the molecular targets being investigated.

Two-photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment.

In this study, a two-photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil-living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two-photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.

Alterations in dietary sodium intake affect cardiovagal baroreflex sensitivity.

High dietary sodium intake has been linked to alterations in neurally mediated cardiovascular function, but the effects of high sodium on cardiovagal baroreflex sensitivity (cBRS) in healthy adults are unknown. The purpose of this study was to determine whether high dietary sodium alters cBRS and heart rate variability (HRV) and whether acute intravenous sodium loading similarly alters cBRS and HRV. High dietary sodium (300 mmol/day, 7 days) was compared with low dietary sodium (20 mmol/day, 7 days; randomized) in 14 participants (38 ± 4 yr old, 23 ± 1 kg/m2 body mass index, 7 women). Acute sodium loading was achieved via a 23-min intravenous hypertonic saline infusion (HSI) in 14 participants (22 ± 1 yr old, 23 ± 1 kg/m2 body mass index, 7 women). During both protocols, participants were supine for 5 min during measurement of beat-to-beat blood pressure (photoplethysmography) and R-R interval (ECG). cBRS was evaluated using the sequence method. Root mean square of successive differences in R-R interval (RMSSD) was used as an index of HRV. Serum sodium (137.4 ± 0.7 vs. 139.9 ± 0.5 meq/l, P < 0.05), plasma osmolality (285 ± 1 vs. 289 ± 1 mosmol/kgH2O, P < 0.05), cBRS (18 ± 2 vs. 26 ± 3 ms/mmHg, P < 0.05), and RMSSD (62 ± 6 vs. 79 ± 10 ms, P < 0.05) were increased following high-sodium diet intake compared with low-sodium diet intake. HSI increased serum sodium (138.1 ± 0.4 vs. 141.1 ± 0.5 meq/l, P < 0.05) and plasma osmolality (286 ± 1 vs. 290 ± 1 mosmol/kgH2O, P < 0.05) but did not change cBRS (26 ± 5 vs. 25 ± 3 ms/mmHg, P = 0.73) and RMSSD (63 ± 9 vs. 63 ± 8 ms, P = 0.99). These data suggest that alterations in dietary sodium intake alter cBRS and HRV but that acute intravenous sodium loading does not alter these indexes of autonomic cardiovascular regulation.

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis.

Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Evidence of the role of the vagal nerves as a monitor in the gastrointestinal-renal axis of natriuresis in human: Effects of vagotomy.

This study aimed to investigate the mechanism of gastrointestinal regulation of natriuresis. Sixteen subjects without (group I) and sixteen subjects with a truncal vagotomy (group II), were given a daily diet of 18mmol of sodium for 5days (D1-D5). The sodium deficit for this period was calculated for each subject and on the morning of day-6 (D6), their cumulative deficit (E) was given as 3% NaCl. In both groups the subjects were divided to receive the hypertonic saline either orally (Ior, IIor) or intravenously (Iiv, IIiv). During the period of low sodium diet when compared to group II subjects of group I (1) had a greater weight loss (p<0.005), (2) demonstrated a larger drop in pulse pressure (p<0.005), (3) achieved a positive sodium equilibrium later (D5 vs D4) and (4) developed a greater sodium deficit (p<0.005). During the two 12h periods of D6, both Ior and Iiv exhibited greater natriuresis during the first 12h period (p<0.0001) whereas both IIor and IIiv did so during the second 12h period (p<0.0001). On D6 Ior excreted the greatest percentage of E (E%; 35.63%±3.12%, p<0.0001) compared to Iiv (17.06%±1.78%), IIor (16.03%±3.54%) and IIiv (15.39%±2.77%) whereas E% was not different between the other subgroups. These results indicate that the differential natriuresis between oral and intravenous sodium loading in previously sodium deprived subjects, is due to a mechanism in which the vagal nerves play a significant role as part of neural reflex or via a natriuretic hormone.

A translational approach for NMDA receptor profiling as a vulnerability biomarker for depression and schizophrenia.

NEW FINDINGS: What is the central question of this study? Can the change in plasma arginine vasopressin concentration (P[AVP] ) in response to osmotic stimulation (POsm ) serve as a biomarker for NMDA receptor signalling in schizophrenia and depression and thereby distinguish between these mental illnesses? What is the main finding and its importance? In response to hyperosmotic challenge, depressed subjects showed increased P[AVP] response compared with healthy control and schizophrenic subjects. However, schizophrenic subjects were not different from healthy control subjects in this small sample. The 'P[AVP] response to POsm ' is a suitable biomarker to distinguish depressed versus schizophrenic patients when used with psychiatric screening. This is the first objective physiological measure for schizophrenia or depression. Altered NMDA receptor activity and glutamate signalling might underlie the pathogenesis of both schizophrenia and depression in subgroups of patients. In schizophrenia, pharmacological modelling, post-mortem and imaging data suggest reduced NMDA signalling. In contrast, recent clinical trials demonstrating the efficacy of the NMDA antagonist ketamine in severely depressed patients suggest increased NMDA receptor signalling. We conducted a proof-of-concept study to assess whether there is any in vivo evidence for an inverse association in depression and schizophrenia with respect to the NMDA receptor function. For this purpose, we used a translational approach, based on findings from animal studies that NMDA receptor is a key mediator of arginine vasopressin (AVP) release into the bloodstream. Using hypertonic saline to increase plasma osmolality (POsm ) and thereby induce AVP release, as done in animal studies, we found that in depressed patients the NMDA receptor-mediated AVP release induced by hypertonic saline infusion was significantly increased [0.24 (0.15) pg ml-1  mosmol-1 , P < 0.05] compared with schizophrenia patients [0.07 (0.07) pg ml-1  mosmol-1 ]. Slopes for healthy control subjects were 0.11 (0.09) pg ml-1  mosmol-1 which was less than the depressed group. These findings are consistent with implicated NMDA receptor-related abnormalities in depression and schizophrenia in subgroups of patients and provide the first in vivo evidence of this dichotomy.

Quantification of nonosmotic sodium storage capacity following acute hypertonic saline infusion in healthy individuals.

The assumption that sodium accumulation in the human body is always accompanied by water retention has been challenged by data showing that sodium can be stored nonosmotically. Here we investigated the contribution of nonosmotic sodium storage to short-term sodium homeostasis after hypertonic saline infusion in healthy individuals on a low-sodium diet. During four hours after infusion, we compared the observed changes in plasma sodium concentration and urinary cation excretion with changes that were calculated with the Adrogue-Madias and Nguyen-Kurtz formula, formulations widely implemented to guide the treatment of dysnatremias. We included 12 healthy non-smoking male individuals with normal blood pressure, body mass index, and kidney function. Right after infusion, the average observed plasma sodium change from baseline (3.5 mmol/L) was similar to the predicted changes by the Adrogue-Madias (3.3 mmol/L) and Nguyen-Kurtz formula (3.1 mmol/L). However, the observed plasma sodium concentration change after four hours (-1.8 mmol/L) was very different from the changes as predicted by the Adrogue-Madias (0.4 mmol/L) and the Nguyen-Kurtz formula (-0.9 mmol/L). Moreover, only 47% and 55%, respectively, of the expected sodium and potassium excretion were retrieved in the urine. Thus, healthy individuals are able to osmotically inactivate significant amounts of sodium after hypertonic saline infusion. Further research is needed to uncover factors that determine nonosmotic sodium storage.

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

5-HT neurons of the area postrema become c-Fos-activated after increases in plasma sodium levels and transmit interoceptive information to the nucleus accumbens.

Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme-producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos-activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium repletion. Non-Tryp-OH neurons also became c-Fos-activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels are expressed in the Tryp-OH-immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP→NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system to the subcortical structures of the reward system. Because these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.

Two non-exclusive strategies employed to protect Torulopsis glabrata against hyperosmotic stress.

Several recent reports described an apoptosis-like programmed cell death (PCD) process in yeast in response to different environmental challenges. In this study, hyperosmotic stress caused by high NaCl concentration in culture medium induced cell death in the haploid yeast Torulopsis glabrata. Propidium iodide (PI) and PI/rhodamine-123 (Rh123) dual staining with flow cytometry showed that high salinity decreased intact cells by 16.5 %, increased necrotic cells by nearly twofold, and altered fermentative parameters appreciably. Morphological and biochemical indicators of apoptosis were apparent, specifically a decrease in mitochondrial membrane potential (∆Ψm), translocation of phosphatidylserine (PS) from the inner to the outer side of the plasma membrane, generation of reactive oxygen species (ROS), and involvement of caspase all while plasma membrane integrity was maintained. Additionally, it was found that overexpression of YCA1 drastically stimulated cell death, indicating that activation of metacaspase might lead to cell death. However, T. glabrata growth under hyperosmotic stress was enhanced when FIS1, HOG1, and GPD2 were overexpressed, or when exogenous proline or glutathione (GSH) were added into the cultures, both of which could repress caspase-3 activity. Thus, in these concrete cases of overexpression of anti-apoptotic or anti-necrotic factors and pharmacological manipulations, it decreased T. glabrata cell death that might help to achieve higher fermentative efficiency.

Diversity and saline resistance of endophytic fungi associated with Pinus thunbergii in coastal shelterbelts of Korea.

The Black Pine, Pinus thunbergii, is widely distributed along the eastern coast of Korea and its importance as a shelterbelt was highlighted after tsunamis in Indonesia and Japan. The root endophytic diversity of P. thunbergii was investigated in three coastal regions; Goseong, Uljin, and Busan. Fungi were isolated from the root tips, and growth rates of pure cultures were measured and compared between PDA with and without 3% NaCl to determine their saline resistance. A total of 259 isolates were divided into 136 morphotypes, of which internal transcribed spacer region sequences identified 58 species. Representatives of each major fungi phylum were present: 44 Ascomycota, 8 Zygomycota, and 6 Basidiomycota. Eighteen species exhibited saline resistance, many of which were Penicillium and Trichoderma species. Shoreline habitats harbored higher saline-tolerant endophytic diversity compared with inland sites. This investigation indicates that endophytes of P. thunbergii living closer to the coast may have higher resistance to salinity and potentially have specific relationships with P. thunbergii.

Estudio sobre la eficacia y utilidad de la solución salina hipertónica al 3 porciento en la bronquiolitis aguda del lactante hospitalizado / Nebulized 3 percent hypertonic saline solution in hospitalized infants with acute bronchiolitis: case-control study on the utility and effectiveness

Objetivo: estudiar la utilidad de la solución salina hipertónica (SSH) al 3% inhalada en el tratamiento de la bronquiolitis aguda (BA) del lactante hospitalizado. Pacientes y métodos: estudio de casos y controles realizado con 639 pacientes de edad inferior a siete meses e ingresados con diagnóstico de BA, primer episodio, durante tres periodos estacionales consecutivos, en la sección de lactantes de un hospital pediátrico de Madrid (España). Los pacientes que recibieron como tratamiento, durante los dos primeros periodos estacionales, suero salino fisiológico (SSF) inhalado con o sin medicación se consideraron el grupo control y los pacientes que recibieron, durante el tercer periodo estacional, suero salino hipertónico al 3% inhalado con o sin medicación se consideraron como casos. Los días de hospitalización y las horas de oxigenoterapia fueron utilizados como medidas de resultado. Resultados: de la totalidad de los niños estudiados, 460 recibieron SSF inhalado, y 179 recibieron SSH al 3%. En el grupo que recibió SSF, la estancia media en el hospital fue de 5,16 días (intervalo de confianza del 95% [IC 95%]: 4,78-5,56) y el tiempo medio de oxigenoterapia fue de 57,34 (IC 95%: 52,93-61,75) frente a 4,90 días (IC 95%: 4,64-5,07) y 67,53 horas (IC 95%: 60,36-74,69), respectivamente, en el grupo tratado con SSH. Estos resultados no alcanzan significación estadística. Los pacientes con estudio positivo de virus respiratorio sincitial (VRS) en aspirado nasofaríngeo y que recibieron SSF necesitaron menos horas de oxígeno de manera significativa (p=0,004), así como aquellos que tenían edad <3 meses (p=0,007). Conclusiones: los resultados obtenidos muestran que la SSH al 3% inhalada no resulta eficaz para reducir la estancia hospitalaria ni el tiempo de oxigenoterapia en los pacientes con BA; además, en los niños menores de tres meses y con estudio positivo de VRS en aspirado nasofaríngeo la aplicación de SSF inhalado consiguió una necesidad menor de horas de oxígeno (AU)

Objective: to study the utility of nebulized 3% hypertonic saline solution (HSS) in hospitalized infants with acute bronchiolitis. Patients and methods: case-control studies accomplished on 639 patients of age less than 7 months old and hospitalized with the diagnosis of acute bronchiolitis, first episode, during 3 consecutive seasons in a pediatric department in Madrid. The patients who received 0.9% saline solution (FSS), with or without medication, during the 2 first seasons were considered the control group and the patients who received, the last season period, nebulized 3% hypertonic saline solution were considered the cases group. The days of hospitalization and the hours of oxygen therapy were used as the result measurement. Results: from the total of the studied children, 460 received 0.9% saline solution and 179 received 3% hypertonic saline solution. In the group receiving FSS the average stay in hospital was 5.16 days (95% confidence interval [95% CI] 4.78-5.56) and the average time of oxygen therapy was 57.34 hours (95% CI 52.93-61.75) opposite to 4.90 days (95% CI 4.64-5.07) and 67.53 hours (95% CI 60.36- 74.69) respectively in the group that received HSS. There was no significant difference between the groups. The patients who received FSS and were positive for VRS and also patients less than 3 months old, showed a significant reduction in the oxygen therapy hours (p= 0.004 and p= 0.007 respectively). Conclusions: results show that 3% hypertonic saline solution has not been effective in reducing hospital stay or length of oxygen therapy in patients with acute bronchiolitis; but nebulized 0,9% saline solution in children with age <3 months and positive study of respiratory syncitial virus in nasopharyngeal aspirate showed a reduced need of hours of oxygen (AU)

Effect of a hypertonic balanced ketone solution on plasma, CSF and brain beta-hydroxybutyrate levels and acid-base status.

PURPOSE: Although glucose is the main source of energy for the human brain, ketones play an important role during starvation or injury. The purpose of our study was to investigate the metabolic effects of a novel hypertonic sodium ketone solution in normal animals. METHODS: Adult Sprague-Dawley rats (420-570 g) were divided into three groups of five, one control and two study arms. The control group received an intravenous infusion of 3 % NaCl at 5 ml/kg/h. The animals in the two study arms were assigned to receive one of the two formulations of ketone solutions, containing hypertonic saline with 40 and 120 mmol/l beta-hydroxybutyrate, respectively. This was infused for 6 h and then the animal was euthanized and brains removed and frozen. RESULTS: Both blood and cerebrospinal fluid (CSF) levels of beta-hydroxybutyrate (BHB) demonstrated strong evidence of a change over time (p < 0.0001). There was also strong evidence of a difference between groups (p < 0.0001). Multiple comparisons showed all these means were statistically different (p < 0.05). Measurement of BHB levels in brain tissue found strong evidence of a difference between groups (p < 0.0001) with control: 0.15 mmol/l (0.01), BHB 40: 0.19 mmol/l (0.01), and BHB 120: 0.28 mmol/l (0.01). Multiple comparisons showed all these means were statistically different (p < 0.05). There were no differences over time (p = 0.31) or between groups (p = 0.33) or an interaction between groups and time (p = 0.47) for base excess. CONCLUSION: The IV infusions of hypertonic saline/BHB are feasible and lead to increased plasma, CSF and brain levels of BHB without significant acid/base effects.

Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress.

Elevated tear osmolarity is one of the key pathological factors in dry eye leading to ocular discomfort associated with damage to the ocular surface and inflammation. The aim of this study was to determine the capacity of the organic osmolyte, betaine, to act as an osmoprotectant against hypertonic stress-induced human corneal epithelial cell shrinkage and apoptosis using in vitro cell culture models. Human corneal limbal epithelial (HCLE) cells exposed to culture medium for 16 h at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) in the presence or absence of betaine (5 or 10 mM) were evaluated for cell volume changes; cell viability; and apoptosis. Betaine (10 mM) ameliorated hyperosmotically induced reduction of cell volume (from 27% reduction to 11%) and resulted in increased mitochondrial activity (by 17%) and an increase in viable cell numbers (by 12%) compared to controls (exposure to hyperosmotic medium without betaine). Hyperosmotically shocked HCLE cells in the presence of betaine (10 mM) halved the number of damaged cells (apoptotic/necrotic) compared to cells in the absence of betaine. The presence of betaine (at 5 or 10 mM) significantly reduced the activity of caspase-8, -9 and -3/7 and release of TNF-α was also reduced by 34% or 55% after exposure of HCLE to 500 mOsm in the presence of 5 or 10 mM betaine, respectively. Using polyclonal antibody against Betaine/GABA transporter 1 (BGT-1), we detected the presence of BGT-1 in HCLE. We demonstrated that the transport of betaine was facilitated by increased osmolarity. In conclusion, betaine stabilized corneal epithelial cell volume under hyperosmotic stress and limited hyperosmotic stress-induced HCLE apoptosis.

Candida famata (Candida flareri).

Candida famata (Candida flareri) belongs to the group of so-called 'flavinogenic yeasts', capable of riboflavin oversynthesis under condition of iron starvation. Some strains of C. famata belong to the most flavinogenic organisms known and were used for industrial production of riboflavin for a long time in the USA. C. famata is characterized by high salt tolerance, growing at NaCl concentrations of up to 2.5 M. Development of basic tools for the metabolic engineering of C. famata, such as a transformation system, selective markers, insertional mutagenesis, a reporter system and others, are described. The developed tools were used for cloning and identification of structural and regulatory genes of riboflavin synthesis. The construction of improved yeast strains producing riboflavin, FMN and FAD, based on the industrial riboflavin-producing strain dep8 and its non-reverting analogue AF4, is also described.

The impact of hypertonic saline inhalation on mucociliary clearance and nasal nitric oxide.

Nitric oxide (NO) has an influence on airway physiology by mediation in ciliary activity, inflammation, host defence, bronchial tone, and pulmonary vascular resistance. However, the clinical relevance of the measurement of nasal NO (nNO) in different physiological and pathological conditions has yet to be established before it can be used as a diagnostic tool. The aim of our study was to establish the relation between nNO and mucociliary clearance (MCC) and the impact of hypertonic saline inhalation on these markers. We examined 43 subjects. nNO was measured in the right (RnNO) and left (LnNO) nostrils before and after inhalation of hypertonic saline (HS) using a NIOX® analyser. MCC was measured before and after inhalation of HS by saccharin test. The levels of nNO before inhalation of HS (RnNO 806 ppb, IQR-337.6; LnNO 854 ppb, IQR 295.8) were significantly lower than levels after inhalation (RnNO 841.8 ppb, IQR 342.3; LnNO 897.4 ppb, IQR 304.1) (p<0.05, Wilcoxon T-test). The difference between RnNO and LnNO before and after inhalation of HS was not statistically significant. However, the difference in MCC before inhalation (507 s, IQR 233) and after inhalation of aerosol (360 s, IQR 238) was statistically significant (p<0.0001, Wilcoxon T-test). We did not confirm a correlation between nNO and MCC. An increase in osmolarity of the airway surface increases MCC and nNO, which could help to clear accumulated secretions in the airways and prevent respiratory tract infections.

Transient opening of the perineurial barrier for analgesic drug delivery.

Selective targeting of sensory or nociceptive neurons in peripheral nerves remains a clinically desirable goal. Delivery of promising analgesic drugs is often impeded by the perineurium, which functions as a diffusion barrier attributable to tight junctions. We used perineurial injection of hypertonic saline as a tool to open the perineurial barrier transiently in rats and elucidated the molecular action principle in mechanistic detail: Hypertonic saline acts via metalloproteinase 9 (MMP9). The noncatalytic hemopexin domain of MMP9 binds to the low-density lipoprotein receptor-related protein-1, triggers phosphorylation of extracellular signal-regulated kinase 1/2, and induces down-regulation of the barrier-forming tight junction protein claudin-1. Perisciatic injection of any component of this pathway, including MMP9 hemopexin domain or claudin-1 siRNA, enables an opioid peptide ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) and a selective sodium channel (NaV1.7)-blocking toxin (ProToxin-II) to exert antinociceptive effects without motor impairment. The latter, as well as the classic TTX, blocked compound action potentials in isolated nerves only after disruption of the perineurial barrier, which, in return, allowed endoneurally released calcitonin gene-related peptide to pass through the nerve sheaths. Our data establish the function and regulation of claudin-1 in the perineurium as the major sealing component, which could be modulated to facilitate drug delivery or, potentially, reseal the barrier under pathological conditions.

Resuscitation with hydroxyethyl starch improves renal function and lactate clearance in penetrating trauma in a randomized controlled study: the FIRST trial (Fluids in Resuscitation of Severe Trauma).

BACKGROUND: The role of fluids in trauma resuscitation is controversial. We compared resuscitation with 0.9% saline vs hydroxyethyl starch, HES 130/0.4, in severe trauma with respect to resuscitation, fluid volume, gastrointestinal recovery, renal function, and blood product requirements. METHODS: Randomized, controlled, double-blind study of severely injured patients requiring >3 litres of fluid resuscitation. Blunt and penetrating trauma were randomized separately. Patients were followed up for 30 days. RESULTS: A total of 115 patients were randomized; of which, 109 were studied. For patients with penetrating trauma (n=67), the mean (sd) fluid requirements were 5.1 (2.7) litres in the HES group and 7.4 (4.3) litres in the saline group (P<0.001). In blunt trauma (n=42), there was no difference in study fluid requirements, but the HES group required significantly more blood products [packed red blood cell volumes 2943 (1628) vs 1473 (1071) ml, P=0.005] and was more severely injured than the saline group (median injury severity score 29.5 vs 18; P=0.01). Haemodynamic data were similar, but, in the penetrating group, plasma lactate concentrations were lower over the first 4 h (P=0.029) and on day 1 with HES than with saline [2.1 (1.4) vs 3.2 (2.2) mmol litre⁻¹; P=0.017]. There was no difference between any groups in time to recovery of bowel function or mortality. In penetrating trauma, renal injury occurred more frequently in the saline group than the HES group (16% vs 0%; P=0.018). In penetrating trauma, maximum sequential organ function scores were lower with HES than with saline (median 2.4 vs 4.5, P=0.012). No differences were seen in safety measures in the blunt trauma patients. CONCLUSIONS: In penetrating trauma, HES provided significantly better lactate clearance and less renal injury than saline. No firm conclusions could be drawn for blunt trauma. STUDY REGISTRATION: ISRCTN 42061860.