The impact of nebulized epoprostenol and iloprost on hemoglobin oxygen affinity: an ex vivo experiment.

Inhalational prostacyclins act as strong vasodilators, potentially improving oxygenation by reducing shunt fraction and ventilation-perfusion mismatch. As prostacyclin receptors are known to be present on human erythrocytes, possible direct effects on hemoglobin oxygen transport were further explored by examining the sole in vitro influence of prostacyclins on hemoglobin oxygen (Hb-O2) affinity. Venous blood samples from 20 healthy volunteers were exposed in vitro to supramaximal doses of epoprostenol, iloprost, and compared with control. By high-throughput measurements, hemoglobin oxygen dissociation curves (ODCs) were derived. Hb-O2 affinity, expressed by P50 and Hill coefficient, was determined and analyzed for three subgroups: males (n = 10), females not taking oral contraceptives (n = 4), and females taking oral contraceptives (n = 6). Epoprostenol significantly decreased P50 in all (males, females without contraceptives, and females taking oral contraceptives) [27.5 (26.4-28.6) mmHg (control) vs. 24.2 (22.7-25.3) mmHg; P < 0.001. median (interquartile range, IQR)] thereby increasing Hb-O2 affinity. Inversely, iloprost only showed significant effects in females taking oral contraceptives where P50 was markedly increased and therefore Hb-O2 affinity decreased [28.4 (27.9-28.9) mmHg (control) vs. 34.4 (32.2-36.0) mmHg; P < 0.001]. Prostacyclin-receptor stimulation and subsequent cAMP-mediated ATP release from erythrocytes are discussed as a possible underlying mechanism for the effect of epoprostenol on Hb-O2 affinity. The reason for the sex hormone-modified iloprost effect remains unclear. Being aware of potentially differing effects on Hb-O2 affinity might help select the right prostacyclin (epoprostenol vs. iloprost) depending on the patient and the underlying disease (e.g., acute respiratory distress syndrome vs. peripheral arterial disease).

Long-term results after posterior open glenoid wedge osteotomy for posterior shoulder instability associated with excessive glenoid retroversion.

BACKGROUND: Treatment of posterior shoulder instability (PSI) associated with excessive glenoid retroversion is a rare, challenging problem in shoulder surgery. One proposed technique is posterior open wedge glenoid osteotomy to correct excessive glenoid retroversion as described by Scott. However, this operation is rarely performed, and limited long-term outcomes using this approach are available. The goal of this study was to analyze the long-term outcomes of posterior open wedge glenoid osteotomy for PSI associated with excessive glenoid retroversion. METHODS: Six consecutive patients (7 shoulders) with a mean age of 24 years (range 19-34) were treated with posterior open wedge glenoid osteotomy for PSI associated with a glenoid retroversion greater than 15° and followed up clinically and radiographically at a mean age of 15 years (range 10-19). RESULTS: Recurrent, symptomatic PSI was observed in 6 of 7 shoulders (86%). One necessitated revision with a posterior (iliac crest) bone block procedure and was rated as a failure and excluded from functional analysis. One patient rated his result as excellent, 3 as good, 1 as fair, and 1 as unsatisfactory. Mean relative Constant Score (CS%) was unchanged from preoperation to final follow-up (CS% = 72%) and pain did not significantly decrease (Constant Score = 7-10 points; P = .969). The mean Subjective Shoulder Value (SSV) improved postoperatively, but with 6 patients the improvement did not reach statistical significance (SSV = 42%-67%, P = .053) and the total Western Ontario Shoulder Instability Index averaged 30% at the final follow-up. Mean glenoid retroversion of all 7 shoulders was corrected from 20° (range 16°-26°) to 3° (range -3° to +8°) (P = .018). In the 5 shoulders with preoperative static posterior subluxation of the humeral head, the humeral head was not recentered. All 7 shoulders showed progression of glenoid arthritic changes. CONCLUSIONS: Posterior open wedge glenoid osteotomy for PSI associated with excessive glenoid retroversion neither reliably restored shoulder stability nor recentered the joint or prevent progression of osteoarthritis. Alternative treatments for PSI associated with excessive glenoid retroversion have to be developed and evaluated.

Stimulation of surfactant exocytosis in primary alveolar type II cells by A. fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen with small airborne spores (conidia) that may escape clearance by upper airways and directly impact the alveolar epithelium. Consequently, innate alveolar defense mechanisms are being activated, including professional phagocytosis by alveolar macrophages, recruitment of circulating neutrophils and probably enhanced secretion of pulmonary surfactant by the alveolar type II (AT II) cells. However, no data are available in support of the latter hypothesis. We therefore used a coculture model of GFP-Aspergillus conidia with primary rat AT II cells and studied fungal growth, cellular Ca2+ homeostasis, and pulmonary surfactant exocytosis by live cell video microscopy. We observed all stages of fungal development, including reversible attachment, binding and internalization of conidia as well as conidial swelling, formation of germ tubes and outgrowth of hyphae. In contrast to resting conidia, which did not provoke immediate cellular effects, metabolically active conidia, fungal cellular extracts (CE) and fungal culture filtrates (CF) prepared from swollen conidia caused a Ca2+-independent exocytosis. Ca2+ signals of greatly varying delays, durations and amplitudes were observed by applying CE or CF obtained from hyphae of A. fumigatus, suggesting compounds secreted by filamentous A. fumigatus that severely interfere with AT II cell Ca2+ homeostasis. The mechanisms underlying the stimulatory effects, with respect to exocytosis and Ca2+ signaling, are unclear and need to be identified.

Posterior Open-wedge Osteotomy and Glenoid Concavity Reconstruction Using an Implant-free, J-shaped Iliac Crest Bone Graft in Atraumatic Posterior Instability with Pathologic Glenoid Retroversion and Dysplasia: A Preliminary Report.

BACKGROUND: Atraumatic posterior shoulder instability in patients with pathologic glenoid retroversion and dysplasia is an unsolved problem in shoulder surgery. QUESTIONS/PURPOSES: In a preliminary study of a small group of patients with atraumatic posterior shoulder instability associated with glenoid retroversion ≥ 15° and glenoid dysplasia who underwent posterior open-wedge osteotomy and glenoid concavity reconstruction using an implant-free, J-shaped iliac crest bone graft, we asked: (1) What proportion of the patients had persistent apprehension? (2) What were the improvements in patient-reported shoulder scores? (3) What were the radiographic findings at short-term follow-up? METHODS: Between 2016 and 2018, we treated seven patients for atraumatic posterior shoulder instability. We performed this intervention when posterior shoulder instability symptoms were unresponsive to physiotherapy for at least 6 months and when it was associated with glenoid retroversion ≥ 15° and dysplasia of the posteroinferior glenoid. All seven patients had a follow-up examination at a minimum of 2 years. The median (range) age at surgery was 27 years (16 to 45) and the median follow-up was 2.3 years (2 to 3). Apprehension was assessed by a positive posterior apprehension and/or posterior jerk test. Patient-reported shoulder scores were obtained and included the subjective shoulder value, obtained by chart review (and scored with 100% representing a normal shoulder; minimum clinically important difference [MCID] 12%), and the Constant pain scale score (with 15 points representing no pain; MCID 1.5 points). Radiographic measurements included glenohumeral arthropathy and posterior humeral head subluxation, bone graft union, correction of glenoid retroversion and glenoid concavity depth, as well as augmentation of glenoid surface area. All endpoints were assessed by individuals not involved in patient care. RESULTS: In four of seven patients, posterior apprehension was positive, but none reported resubluxation. The preoperative subjective shoulder value (median [range] 40% [30% to 80%]) and Constant pain scale score (median 7 points [3 to 13]) were improved at latest follow-up (median subjective shoulder value 90% [70% to 100%]; p = 0.02; median Constant pain scale score 15 points [10 to 15]; p = 0.03). Posterior glenoid cartilage erosion was present in four patients (all four had Walch Type B1 glenoids) preoperatively and showed no progression until the final follow-up examination. The median (range) humeral head subluxation index decreased from 69% (54% to 85%) preoperatively to 55% (46% to 67%) postoperatively (p = 0.02), and in two of four patients with preoperative humeral head subluxation (> 65% subluxation), it was reversed to a centered humeral head. CT images showed union in all implant-free, J-shaped iliac crest bone grafts. The median preoperative retroversion was corrected from 16° (15° to 25°) to 0° postoperatively (-5° to 6°; p = 0.02), the median glenoid concavity depth was reconstructed from 0.3 mm (-0.7 to 1.6) preoperatively to 1.2 mm (1.1 to 3.1) postoperatively (p = 0.02), and the median preoperative glenoid surface area was increased by 20% (p = 0.02). No intraoperative or postoperative complications were recorded, and no reoperation was performed or is planned. CONCLUSION: In this small, retrospective series of patients treated by experienced shoulder surgeons, a posterior J-bone graft procedure was able to reconstruct posterior glenoid morphology, correct glenoid retroversion, and improve posterior shoulder instability associated with pathologic glenoid retroversion and dysplasia, although four of seven patients had persistent posterior apprehension. Although no patients in this small series experienced complications, the size and complexity of this procedure make it likely that as more patients have it, some will develop complications; future studies will need to characterize the frequency and severity of those complications, and we recommend that this procedure be done only by experienced shoulder surgeons. The early results in these seven patients justify further study of this procedure for the proposed indication, but longer term follow-up is necessary to continue to assess whether it is advantageous to combine the reconstruction of posterior glenoid concavity with correction of pathological glenoid retroversion and increasing glenoid surface compared with traditional surgical techniques such as the posterior opening wedge osteotomy or simple posterior bone block procedures. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Consistency of 3D femoral torsion measurement from MRI compared to CT gold standard.

BACKGROUND: Several hip and knee pathologies are associated with aberrant femoral torsion. Diagnostic workup includes computed tomography (CT) and magnetic resonance imaging (MRI). For three-dimensional (3D) analysis of complex deformities it would be desirable to measure femoral torsion from MRI data to avoid ionizing radiation of CT in a young patient population. 3D measurement of femoral torsion from MRI has not yet been compared to measurements from CT images. We hypothesize that agreement will exist between MRI and CT 3D measurements of femoral torsion. METHODS: CT and MRI data from 29 hips of 15 patients with routine diagnostic workup for suspected femoroacetabular impingement (FAI) were used to generate 3D bone models. 3D measurement of femoral torsion was performed by two independent readers using the method of Kim et al. which is validated for CT. Inter-modalitiy and inter-reader intraclass correlation coefficients (ICC) were calculated. RESULTS: Between MRI and CT 3D measurements an ICC of 0.950 (0.898; 0.976) (reader 1) respectively 0.950 (0.897; 0.976) (Reader 2) was found. The ICC (95% CI) expressing the inter-reader reliability for both modalities was 0.945 (0.886; 0.973) for MRI and 0.957 (0.910; 0.979) for CT, respectively. Mean difference between CT and MRI measurement was 0.42° (MRI - CT, SD: 2.77°, p = 0.253). CONCLUSIONS: There was consistency between 3D measurements of femoral torsion between computer rendered MRI images compared to measurements with the "gold standard" of CT images. ICC for inter-modality and inter-reader consistency indicate excellent reliability. Accurate, reliable and reproducible 3D measurement of femoral torsion is possible from MRI images.

Occult periprosthetic fractures of the acetabulum in THA using an elliptic cup design have no adverse impact on outcome.

Occult periprosthetic fractures have been defined as a fracture only visible on postoperative CT scans but not on postoperative plain radiography after an uneventful surgery without intraoperative fracture. The fracture rate for hemispherical and peripheral self-locking cups has been described as 8.4%. We retrospectively analyzed postoperative CT scans after primary THA to clear the question whether such occult periprosthetic fractures of the acetabulum require particular treatment strategy. Between 2014 and 2018 we identified 115 CT scans of 114 patients after primary cementless THA with elliptical cups using a direct anterior approach. The CT scans were obtained as part of other investigations. Localization of the fracture, patients demographics, clinical (WOMAC, Harris Hip Score) and radiological outcome were analyzed. Fracture and non-Fracture group were compared with regard to demographics and short-term outcome after 1 year. Four occult fractures (3.5%) were identified. Three fractures involved the posterior wall. All patients had an uneventful routine postoperative rehabilitation. Patients with occult fractures showed similar post- operative HHS and WOMAC scores at 3 (HHS p = 0.576, WOMAC p = 0.128) and 12 (HHS p = 0.479, WOMAC p = 0.588) months. There were no cup loosening nor radiolucent lines at latest follow-up (mean FU 22 months, range 12-34 months). Clinical and radiological short-term outcome was not impaired by the occurrence of an occult periprosthetic fracture of the acetabulum. The incidental detection of an occult periprosthetic fracture of the acetabulum does not seem to oblige the surgeon to adapt the postoperative regime.

The impact of mal-angulated femoral rotational osteotomies on mechanical leg axis: a computer simulation model.

BACKGROUND: Subtrochanteric or supracondylar femoral rotational osteotomies are established surgical treatments for femoral rotational deformities. Unintended change of the mechanical leg axis is an identified problem. Different attempts exist to plan a correct osteotomy plane, but implementation of the preoperative planning into the surgical situation can be challenging. Goal of this study was to identify the critical threshold of mal-angulation of the osteotomy plane and of femoral rotation that leads to a relevant deviation of the postoperative mechanical leg axis using a computer simulation approach. METHODS: Three-dimensional (3D) surface models of the lower extremity of two patients (Model 1: 42° femoral antetorsion; Model 2: 6° femoral retrotorsion) were generated from computed tomography data. First, baseline subtrochanteric and supracondylar rotational osteotomies, perpendicular to the femoral mechanical axis were simulated. Afterwards, mal-angulated osteotomies in sagittal and frontal plane followed by different degrees of rotation were simulated and frontal mechanical axis was analyzed. RESULTS: 400 mal-angulated osteotomies have been simulated. Mal-angulation of ±30° with 30° rotation showed maximum deviation from preoperative mechanical axis in subtrochanteric osteotomies (4.0° ± 0.4°) and in supracondylar osteotomies (12.4° ± 0.8°). Minimal mal-angulation of 15° in sagittal plane in subtrochanteric osteotomies and mal-angulation of 10° in sagittal plane in supracondylar osteotomies altered the mechanical axis by > 2°. Mal-angulation in sagittal plane showed higher deviations of the mechanical axis (up to 12.4° ± 0.8°), than in frontal plane mal-angulation (up to 4.0° ± 1.9°). CONCLUSION: A femoral rotational osteotomy, perpendicular to the femoral mechanical axis, has no considerable influence on the mechanical leg axis. However, mal-angulation of femoral rotational osteotomies showed relevant changes of the mechanical leg axis. In supracondylar respectively subtrochanteric procedures, mal-angulation of only 10° in combination with already 15° of femoral rotation respectively mal-angulation of 15° in combination with 30° of femoral rotation, can lead to a relevant postoperative mechanical leg axis deviation of more than 2°, wherefore these patients probably would benefit from the use of navigation aids.

Outcome of Ray Resection as Definitive Treatment in Forefoot Infection or Ischemia: A Cohort Study.

Ray resection is frequently performed in cases of infection or ischemia, but the literature is scarce concerning its outcome as a definitive treatment. In this retrospective cohort study, we reviewed our cohort with transmetatarsal ray resection with a mean follow-up of 36.3 months. Reulcerations, transfer ulcers, and reamputations were determined. Risk factor analysis for revision surgery was conducted. Among 185 patients, 71 (38.4%) had revision surgery within a mean of 1.4 ± 2.6 years (range 2 days to 12.9 years), 22 (11.9%) had major amputations, 49 (26.5%) had minor amputations, 11 (5.9%) had same-ray reulceration, 40 (21.6%) had transfer ulceration, and 2 (1.1%) had both reulceration and transfer ulceration. Occurrence of a postoperative ulcer was statistically significantly associated with revision surgery (p < .01). In conclusion, metatarsal ray resection is a reasonable treatment option in cases of forefoot ischemia or infection to prevent major amputation but fails in 11.9%, and reulceration is associated with further revisions, making ulcer prevention paramount.

Revision After Total Transmetatarsal Amputation.

Total transmetatarsal amputation (TMA) can be an option for foot salvage in gangrene, sepsis, or infected necrosis. However, the literature concerning predictive outcome factors and bacterial sampling is scarce. To identify potential associations between revision surgery and underlying bacteria or other preoperative selection criteria, we reviewed all patients with TMA who were treated at our institution. We compared the patients with remissions with surgical revisions. Among 96 adult patients with TMA (105 amputations), 42 required a revision surgery (40%), 18 had a further minor proximal surgical reamputation (17%) and 18 had a major proximal surgical reamputation (14%). In group comparisons, a previous infection with Staphylococcus aureus was protective with a lower revision risk (4/26 with revision surgery vs 22/26 without revisions; p = .03). This was the opposite for postoperative persistent soft tissue or bone infections (p < .01) and delayed wound healing (p < .01), which were positively associated with a revision risk. The American Society of Anesthesiologists Score, sex, age, body mass index, diabetes, polyneuropathy, chronic renal failure, dialysis, peripheral arterial disease, smoking status, and antibiotic regimen did not influence this revision risk. These results must be interpreted cautiously because no multiple variable calculations could be conducted as a result of the paucity of cases and confounding could not be evaluated sufficiently. TMA is an option to prevent major amputations, but it may be associated with a subsequent revision risk of 40% in adult patients. In our cohort study, persistent postamputation infection and delayed wound healing were associated with revision. However, no preoperative selection criteria were found that lead to revision surgery except for an infection with Staphylococcus aureus, which protected against revision surgery.

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes.

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.

A small key unlocks a heavy door: The essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

Pneumocytes Assemble Lung Surfactant as Highly Packed/Dehydrated States with Optimal Surface Activity.

Pulmonary surfactant (PS) is an essential complex of lipids and specific proteins synthesized in alveolar type II pneumocytes, where it is assembled and stored intracellularly as multilayered organelles known as lamellar bodies (LBs). Once secreted upon physiological stimulation, LBs maintain a densely packed structure in the form of lamellar body-like particles (LBPs), which are efficiently transferred into the alveolar air-water interface, lowering surface tension to avoid lung collapse at end-expiration. In this work, the structural organization of membranes in LBs and LBPs freshly secreted by primary cultures of rat ATII cells has been compared with that of native lung surfactant membranes isolated from porcine bronchoalveolar lavage. PS assembles in LBs as crystalline-like highly ordered structures, with a highly packed and dehydrated state, which is maintained at supraphysiological temperatures. This relatively ordered/packed state is retained in secreted LBPs. The micro- and nanostructural examination of LBPs suggests the existence of high levels of structural complexity in comparison with the material purified from lavages, which may contain partially inactivated or spent structures. Additionally, freshly secreted surfactant LBPs exhibit superior activity when generating interfacial films and a higher intrinsic resistance to inactivating agents, such as serum proteins or meconium. We propose that LBs are assembled as an energy-activated structure competent to form very efficient interfacial films, and that the organization of lipids and proteins and the properties displayed by the films formed by LBPs are likely similar to those established at the alveolar interface and represent the actual functional structure of surfactant as it sustains respiration.

Physiological variables affecting surface film formation by native lamellar body-like pulmonary surfactant particles.

Pulmonary surfactant (PS) is a surface active complex of lipids and proteins that prevents the alveolar structures from collapsing and reduces the work of breathing by lowering the surface tension at the alveolar air-liquid interface (ALI). Surfactant is synthesized by the alveolar type II (AT II) cells, and it is stored in specialized organelles, the lamellar bodies (LBs), as tightly packed lipid bilayers. Upon secretion into the alveolar lining fluid, a large fraction of these particles retain most of their packed lamellar structure, giving rise to the term lamellar body like-particles (LBPs). Due to their stability in aqueous media, freshly secreted LBPs can be harvested from AT II cell preparations. However, when LBPs get in contact with an ALI, they quickly and spontaneously adsorb into a highly organized surface film. In the present study we investigated the adsorptive capacity of LBPs at an ALI under relevant physiological parameters that characterize the alveolar environment in homeostatic or in pathological conditions. Adsorption of LBPs at an ALI is highly sensitive to pH, temperature and albumin concentration and to a relatively lesser extent to changes in osmolarity or Ca(2+) concentrations in the physiological range. Furthermore, proteolysis of LBPs significantly decreases their adsorptive capacity confirming the important role of surfactant proteins in the formation of surface active films.

Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes.

Ca(2+) is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca(2+) concentration ([Ca(2+)])(c) leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca(2+) channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca(2+) entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca(2+) entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca(2+) channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X(4) receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X(4) receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X(4). Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca(2+) influx via vesicular Ca(2+) channels regulates fusion pore expansion and vesicle content release.

The impact of cannabinoids on methemoglobin formation and hemoglobin oxygen affinity: An ex-vivo study.

The affinity of hemoglobin (Hb) to oxygen (O2) influences processes of oxygen delivery and extraction at the tissue level. Despite cannabinoids being utilized or ingested in various ways, their possible impact on Hb-O2 affinity has barely been studied. This is an experimental ex-vivo trial. Venous blood samples were drawn from 5 male and 6 female healthy volunteers and subsequently exposed to different cannabinoid types: (delta-9-tetrahydrocannabinol [Δ9-THC], delta-8-tetrahydrocannabinol [Δ8-THC], cannabidiol [CBD]) at different concentrations. Oxygen dissociation curves (ODC) were measured and blood gas analyses were performed for methemoglobin (MetHb) determination. The results revealed no MetHb formation. Besides two statistically significant changes (+1.4 mmHg and -0.9 mmHg) in the female cohort, following Δ9-THC and Δ8-THC exposure, no further P50 changes could be observed. The study demonstrated an in-vitro effect of selected cannabinoids and dosages on P50 values in female participants, with variations not observed at other dosages, leaving the underlying mechanisms open for debate. MetHb formation, as potential mechanism, was not detected in this study. The precise reasons why changes only occurred at specific dosages remain unclear, indicating a need for further in-vivo research to understand the interaction between cannabinoids and Hb-O2 affinity completely.

Interfacial stress affects rat alveolar type II cell signaling and gene expression.

Previous work from our group (Ravasio A, Hobi N, Bertocchi C, Jesacher A, Dietl P, Haller T. Am J Physiol Cell Physiol 300: C1456-C1465, 2011.) showed that contact of alveolar epithelial type II cells with an air-liquid interface (I(AL)) leads to a paradoxical situation. It is a potential threat that can cause cell injury, but also a Ca(2+)-dependent stimulus for surfactant secretion. Both events can be explained by the impact of interfacial tensile forces on cellular structures. Here, the strength of this mechanical stimulus became also apparent in microarray studies by a rapid and significant change on the transcriptional level. Cells challenged with an I(AL) in two different ways showed activation/inactivation of cellular pathways involved in stress response and defense, and a detailed Pubmatrix search identified genes associated with several lung diseases and injuries. Altogether, they suggest a close relationship of interfacial stress sensation with current models in alveolar micromechanics. Further similarities between I(AL) and cell stretch were found with respect to the underlying signaling events. The source of Ca(2+) was extracellular, and the transmembrane Ca(2+) entry pathway suggests the involvement of a mechanosensitive channel. We conclude that alveolar type II cells, due to their location and morphology, are specific sensors of the I(AL), but largely protected from interfacial stress by surfactant release.

The effect of desflurane, isoflurane and sevoflurane on the hemoglobin oxygen dissociation curve in human blood samples.

Desflurane, isoflurane and sevoflurane, three halogenated ethers, are commonly used inhaled anesthetics, both in the operating room and in the intensive care unit (ICU). The potency and dosage of these drugs is expressed by the MAC value (minimum alveolar concentration). Their interaction with hemoglobin and its affinity for oxygen, best described by the oxygen dissociation curve (ODC), has already been investigated, with conflicting results. Altered by many factors, the ODC can be shifted to the left or to the right, therefore increasing or decreasing hemoglobin oxygen (Hb-O2) affinity. In venous blood samples of 22 healthy participants (11 female, 11 male) ODC were measured with a high-throughput method in vitro. Blood samples were either exposed to control or to three different concentrations of desflurane, isoflurane or sevoflurane prior to and during measurements (low, medium and high corresponding to MAC 0.5, MAC 1.0 and MAC 2.0). With increasing concentrations from control to medium, desflurane and isoflurane significantly decreased Hb-O2 affinity by shifting the ODC to the right (p = 0.016 and p < 0.001) but sevoflurane showed no effects. When further increasing concentrations from medium to high, all three inhaled anesthetics shifted the ODC back to the left (p < 0.001). Comparing only controls to high concentrations, a significant increase in Hb-O2 affinity for desflurane (p = 0.005) and sevoflurane (p < 0.001) was detected. Our study shows a varying effect at different doses of inhaled anesthetics on Hb-O2 affinity. While the underlying mechanisms remain unclear, these results show an effect which needs to be further investigated to determine if patients undergoing anesthesia may potentially benefit or get disadvantage from this slightly increased (e.g. impaired pulmonary oxygen uptake), or decreased Hb-O2 affinity (e.g. arterial vascular disease).Trial registration: This study is registered with clinicaltrials.gov (NCT04612270).

Interfacial sensing by alveolar type II cells: a new concept in lung physiology?

Alveolar type II (AT II) cells are in close contact with an air-liquid interface (I(AL)). This contact may be of considerable physiological relevance; however, no data exist to provide a satisfying description of this specific microenvironment. This is mainly due to the experimental difficulty to manipulate and analyze cell-air contacts in a specific way. Therefore, we designed assays to quantify cell viability, Ca(2+) changes, and exocytosis in the course of interface contact and miniaturized I(AL) devices for direct, subcellular, and real-time analyses of cell-interface interactions by fluorescence microscopy or interferometry. The studies demonstrated that the sole presence of an I(AL) is not sensed by the cells. However, when AT II cells are forced into closer contact with it, they respond promptly with sustained Ca(2+) signals and surfactant exocytosis before the occurrence of irreversible cell damage. This points to a paradoxical situation: a potential threat and potent stimulus for the cells. Furthermore, we found that the signalling mechanism underlying sensation of an I(AL) can be sufficiently explained by mechanical forces. These results demonstrate that the I(AL) itself can play a major, although so-far neglected, role in lung physiology, particularly in the regulatory mechanisms related with surfactant homeostasis. Moreover, they also support a general new concept of mechanosensation in the lung.

Maximum likelihood based classification of electron tomographic data.

Classification and averaging of sub-tomograms can improve the fidelity and resolution of structures obtained by electron tomography. Here we present a three-dimensional (3D) maximum likelihood algorithm--MLTOMO--which is characterized by integrating 3D alignment and classification into a single, unified processing step. The novelty of our approach lies in the way we calculate the probability of observing an individual sub-tomogram for a given reference structure. We assume that the reference structure is affected by a 'compound wedge', resulting from the summation of many individual missing wedges in distinct orientations. The distance metric underlying our probability calculations effectively down-weights Fourier components that are observed less frequently. Simulations demonstrate that MLTOMO clearly outperforms the 'constrained correlation' approach and has advantages over existing approaches in cases where the sub-tomograms adopt preferred orientations. Application of our approach to cryo-electron tomographic data of ice-embedded thermosomes revealed distinct conformations that are in good agreement with results obtained by previous single particle studies.

Lamellar bodies form solid three-dimensional films at the respiratory air-liquid interface.

Pulmonary surfactant is essential for lung function. It is assembled, stored and secreted as particulate entities (lamellar body-like particles; LBPs). LBPs disintegrate when they contact an air-liquid interface, leading to an instantaneous spreading of material and a decline in surface tension. Here, we demonstrate that the film formed by the adsorbed material spontaneously segregate into distinct ordered and disordered lipid phase regions under unprecedented near-physiological conditions and, unlike natural surfactant purified from bronchoalveolar lavages, dynamically reorganized into highly viscous multilayer domains with complex three-dimensional topographies. Multilayer domains, in coexistence with liquid phases, showed a progressive stiffening and finally solidification, probably driven by a self-driven disassembly of LBPs from a sub-surface compartment. We conclude that surface film formation from LBPs is a highly dynamic and complex process, leading to a more elaborated scenario than that observed and predicted by models using reconstituted, lavaged, or fractionated preparations.