Complement C5a-Induced Changes in Neutrophil Morphology During Inflammation.

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies.

Percutaneous left atrial appendage occlusion for stroke prevention in patients with atrial fibrillation and contraindication for anticoagulation.

BACKGROUND: Stroke, as the third cause of death in developed countries, is a public health issue. Atrial fibrillation is an important cause of ischemic stroke and its prevention is efficient with oral anticoagulation. However, oral anticoagulation can be contraindicated because of hemorrhagic risk related to these treatments. Percutaneous left atrial appendage occlusion is a new alternative of oral anticoagulation for patients with atrial fibrillation and high risk of cardio-embolic stroke but contraindicated for oral anticoagulation. METHODS: We describe in this paper the procedure of left atrial appendage occlusion with the Amplatzer cardiac plug device, used in our center in Grenoble university hospital, for the first three patients who have been treated with this device. These three patients (one man and two women) have all atrial fibrillation with neurological complication of this arrhythmia, as ischemic stroke. Oral anticoagulation is indicated to prevent another ischemic stroke. However, they all have a high risk of cerebral bleeding for different reasons (cavernomatosis, history of intracerebral hemorrhage and aneurysm of the polygon of Willis). Consequently, they have a high risk of cardio-embolic complication but contraindication for oral anticoagulation. They have been treated by left atrial appendage occlusion with Amplatzer cardiac plug device by percutaneous and trans-septal access. Then, they have been followed by neurologist and cardiologist, with clinical and paraclinical evaluation by echocardiography. RESULTS: Our three first patients have been successfully implanted, without periprocedural complication. No latest adverse event was observed, and particularly no cardiac or neurologic adverse event. CONCLUSION: The technique of left atrial appendage occlusion is a very interesting and promising technique for ischemic stroke prevention in patient with high risk of cardio-embolic complication because of atrial fibrillation, but high risk of bleeding and contraindication for oral anticoagulation. Because of frequency of both atrial fibrillation and contraindication for oral anticoagulation, occlusion of the left atrial appendage should become an interesting alternative for many patients. However, it remains an invasive procedure and efficacy and indications need to be evaluated in further clinical trials. Risk/benefit ratio must be carefully assessed and compared to that of the new anticoagulant drugs.

[Potential effect of the stimulus threshold level of the nociceptive flexion reflex (NFRT) on mortality and delirium incidence in the critically ill patient: a retrospective cohort analysis]. / Potenzieller Einfluss der Reizschwellenhöhe des nozizeptiven Flexorenreflex (NFRT) auf die Mortalitäts- und Delirinzidenz beim kritisch kranken Patienten: eine retrospektive Kohortenanalyse.

BACKGROUND: Mortality and delirium in critically ill patients are affected by the provided analgesics and sedatives. The deeper the sedation and the higher the dose of analgesics applied, the more difficult it is to assess pain and the depth of sedation. Therefore, instrumental measurement methods, such as the measurement of the stimulus threshold of the nociceptive flexion reflex (NFRT), are becoming increasingly more important. OBJECTIVE: The aim of the present study is to investigate a potential association between the level of the nociceptive flexion reflex, mortality, and the occurrence of delirium. MATERIAL AND METHODS: By retrospectively analyzing a pilot data set of 57 ICU patients from the interdisciplinary surgical ICU of Ulm University Hospital surveyed between 11/2018 and 03/2020, a possible association between the NFRT, mortality, and the occurrence of delirium was calculated in an adjusted logistic regression model. Depending on the cut-off value, the stimulus threshold corridors result in the following comparison pairs: < 20 mA vs. 20-40 mA/20-50 mA/20-60 mA, > 40 mA vs. 20-40 mA, > 50 mA vs. 20-50 mA and > 60 mA vs. 20-60 mA. Results are presented as odds ratios (OR) adjusted for age, sex, height, TISS-28, SAPS II, RASS, BPS, and applied analgesics. Pain assessment was performed, in addition to the Behavioral Pain scale, ≥ 3 times daily by measuring NFRT. RESULTS: A statistically nonsignificant tendency for an increase in mortality incidence occurred with an NFRT > 50 mA, versus a stimulus threshold corridor of 20-50 mA (OR 3.3, CI: 0.89-12.43, p = 0.07). A trend toward a reduction in delirium incidence occurred at an NFRT < 20 mA, versus a stimulus threshold corridor of 20-40 mA (OR 0.40, CI: 0.18-0.92, p = 0.03). CONCLUSION: Based on the level of the NFRT, no recommendation can be made at this point to adjust the analgesic regimen of critically ill patients, who are unable to communicate. The observation of a tendency towards an increase in mortality at high stimulus thresholds or a reduction in the occurrence of delirium at low stimulus thresholds of the NFRT must be verified in standardized studies.

Targeting Epilepsy Through the Foremen Ovale: How Many Helical Needles are Needed?

Laser ablation of the hippocampus offers medically refractory epilepsy patients an alternative to invasive surgeries. Emerging commercial solutions deliver the ablator through a burr hole in the back of the head. We recently introduced a new access path through the foremen ovale, using a helical needle, which minimizes the amount of healthy brain tissue the needle must pass through on its way to the hippocampus, and also enables the needle to follow the medial axis of the hippocampus more closely. In this paper, we investigate whether helical needles should be designed and fabricated on a patient-specific basis as we had previously proposed, or whether a small collection of pre-defined needle shapes can apply across many patients. We propose a new optimization strategy to determine this needle set using patient data, and investigate the accuracy with which these needles can reach the the medial axis of the hippocampus. We find that three basic tube shapes (mirrored as necessary for left vs. right hippocampi) are all that is required, across 20 patient datasets (obtained from 10 patient CT scans), to reduce worst-case maximum error below 2 mm.

Development of a PET/EPRI combined imaging system for assessing tumor hypoxia.

Precise quantitative delineation of tumor hypoxia is essential in radiation therapy treatment planning to improve the treatment efficacy by targeting hypoxic sub-volumes. We developed a combined imaging system of positron emission tomography (PET) and electron para-magnetic resonance imaging (EPRI) of molecular oxygen to investigate the accuracy of PET imaging in assessing tumor hypoxia. The PET/EPRI combined imaging system aims to use EPRI to precisely measure the oxygen partial pressure in tissues. This will evaluate the validity of PET hypoxic tumor imaging by (near) simultaneously acquired EPRI as ground truth. The combined imaging system was constructed by integrating a small animal PET scanner (inner ring diameter 62 mm and axial field of view 25.6 mm) and an EPRI subsystem (field strength 25 mT and resonant frequency 700 MHz). The compatibility between the PET and EPRI subsystems were tested with both phantom and animal imaging. Hypoxic imaging on a tumor mouse model using 18F-fluoromisonidazole radio-tracer was conducted with the developed PET/EPRI system. We report the development and initial imaging results obtained from the PET/EPRI combined imaging system.

Regulation of nerve growth factor synthesis and release in organ cultures of rat iris.

We studied the synthesis and release of nerve growth factor (NGF) in cultured rat iris with a two-site enzyme immunoassay by measuring the time course of NGF levels remaining in the iris and relased into the medium up to 72 h. For up to 3 h, the NGF levels in the iris did not change significantly. After that, they increased to a maximal level of 350 +/- 30 pg NGF/iris at 19 h, which is 200 times higher than the in vivo content. Between 20 and 72 h in culture, the NGF level decreased to 130 +/- 10 pg NGF/iris, whereas general protein synthesis did not change during that time period. Maximal rate of NGF production (203 pg NGF/h/iris) was seen between 9 and 12 h in culture. In the medium, NGF levels were first detectable after 6 h. Levels then increased with a time course similar to that seen within the iris, reaching a maximal level of 1,180 +/- 180 pg after 19 h in vitro, and then did not significantly change for up to 48 h. The NGF production of the densely sympathetically innervated dilator was three times higher than that of the predominantly cholinergically innervated sphincter. The NGF production was blocked by inhibitors of messenger RNA synthesis (actinomycin D) and of polyadenylation (9-beta-D-arabinofuranosyladenine) as well as by inhibitors of translation (cycloheximide). Monensin, which interferes with the transport of proteins through the Golgi apparatus, decreased NGF levels to 8-12% of controls in the medium, suggesting that the Golgi apparatus is involved in the intracellular processing of NGF.

Mesenchymal stem cells in peripheral blood of severely injured patients.

PURPOSE: Mesenchymal stem cells (MSCs) are primarily stromal cells present in bone marrow and other tissues that are crucial for tissue regeneration and can be mobilized into peripheral blood after different types of organ damage. However, little is known about MSC appearance in blood in the setting of polytrauma. METHODS: We conducted a monocentered and longitudinal observational clinical study in 11 polytraumatized patients with an injury severity score (ISS) ≥ 24 to determine the numbers of MSCs in peripheral blood. Blood was collected from healthy volunteers and patients after polytrauma in the emergency room and 4, 12, 24, 48 h, 5 and 10 day later, and cells carrying MSC-surface markers (negative for CD45, positive for CD29, CD73, CD90, CD105, and CD166 in different combinations also employing the more stringent markers STRO1 and MSCA1) were detected and characterized using flow cytometry. Relative numbers of MSC-like cells were correlated with clinical parameters to evaluate if specific injury patterns had an influence on their presence in the blood cell pool. RESULTS: We were able to detect MSC marker-positive cells in both cohorts; however, the percentage of those cells present in the blood of patients during the first 10 day after injury was mostly similar to healthy volunteers, and significantly lowers starting at 4 h post trauma for one marker combination when compared to controls. Furthermore, the presence of a pelvis fracture was partly correlated with reduced relative numbers of MSC-like cells detectable in blood. CONCLUSIONS: Polytrauma in humans was associated with partly reduced relative numbers of MSC-like cells detected in peripheral blood in the time course after injury. Further studies need to define if this reduction was due to lower mobilization from the bone marrow or to active migration to the sites of injury.

Biological screening of extracts from leaf and stem bark of Croton floribundus Spreng. (Euphorbiaceae).

This work describes the preliminary evaluation of cytotoxic, antimicrobial, molluscicidal, antioxidant and anticholinesterase activities from leaf (LECF) and stem bark alcoholic extracts (BECF) of the species Croton floribundus Spreng. (Euphorbiaceae), popularly known as capixingui or tapixingui. BECF presented significant toxicity (LC50 = 89.6 µg/ml) in the Artemia salina Leach, 1819 (Crustacea: Branchiopoda) bioassay, whereas LECF did not show activity (LC50 > 1000 µg/ml). From DPPH method, the values of IC50 for the LECF and BECF were 61.2 µg/ml and 62.2 µg/ml, respectively, showing that C. floribundus has an expressive antioxidant activity. Antimicrobial susceptibility was evaluated by microdilution technique and only BECF was active against Staphylococcus aureus (MIC = 39.6 µg/ml). The extracts did not present molluscicidal activity against snail Biomphalaria glabrata Say, 1818 (Gastropoda: Planorbidae). Both extracts revealed the presence of several components with an inhibiting capacity of acetylcholinesterase enzyme on the bioautographic assay. C. floribundus showed to be a promising species considering that it exhibited good biological activity in the most assays performed.

The use of a new 3D splint and double CT scan procedure to obtain an accurate anatomic virtual augmented model of the skull.

Three-dimensional (3D) virtual planning of orthognathic surgery requires detailed visualization of the interocclusal relationship. The purpose of this study was to introduce the modification of the double computed tomography (CT) scan procedure using a newly designed 3D splint in order to obtain a detailed anatomic 3D virtual augmented model of the skull. A total of 10 dry adult human cadaver skulls were used to evaluate the accuracy of the automatic rigid registration method for fusion of both CT datasets (Maxilim, version 1.3.0). The overall mean registration error was 0.1355+/-0.0323 mm (range 0.0760-0.1782 mm). Analysis of variance showed a registration method error of 0.0564 mm (P < 0.001; 95% confidence interval = 0.0491-0.0622). The combination of the newly designed 3D splint with the double CT scan procedure allowed accurate registration and the set-up of an accurate anatomic 3D virtual augmented model of the skull with detailed dental surface.

A new method of monitoring osteomyelitis.

Chronic infections of bone such as osteomyelitis are frequent events, especially in immunocompromised or diabetic patients, and costly on a national level. Incorrect treatment or delayed diagnosis may lead to loss of the affected extremity or mandible. The aim of this study was to assess the possible value of urinary lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) concentrations in the monitoring of mandibular osteomyelitis. Patients were assigned to the following groups: group 1 (n=85), control; group 2a (n=38), patients with active disease; group 2b (n=25), patients of group 2a 6 months after successful treatment; group 2c (n=7), patients of group 2a with ongoing osteomyelitis 6 months after treatment. The range and upper limit of normal values (HP(max) and LP(max)) were determined in group 1. Levels of LP and HP were measured by high-performance liquid chromatography and fluorescence detection. There was a significant decrease (mean 45.43% for HP and 32.12% for LP) in samples of group 2b compared to 2a (P<0.001 for HP and LP). There was a significant increase in HP values in samples from group 2c compared to 2a (P=0.018). The urinary concentrations of HP and LP appear to act as a marker of disease activity, with a decrease reflecting treatment success and an increase or stable values indicating persistent disease. An inexpensive tool (US$5 per analysis) for the monitoring of osteomyelitis is described.

Evaluation of [Formula: see text] enrichment values obtained with an oral breath test under conditions of impaired gastric functioning.

Gastric emptying can be assessed by an oral administration of a 13C labeled substrate and its response in the expiratory release of the oxidation product [Formula: see text]. Impaired gut function, reflected, for example, in an intolerance against enteral nutrition may delay or discontinue gastric emptying, potentially leading to multiple peaks in the time profile of expiration. The resulting profile cannot be analyzed by the usual data evaluation that is based on a 'beta exponential' (BEX) function. We developed a new approach that better reflects the underlying physiology. It allows a flexible time profile of gastric release and considers a transient [Formula: see text] retention in different compartments as well as an incomplete recovery of [Formula: see text] in the expiration. Parameters that describe the distribution/retention kinetics cannot be determined based on the same breath data that were used to estimate emptying. To enable the determination of the kinetic parameters, they were constrained to match published data using a Bayesian statistical analysis. The applicability of the new model was compared with BEX for healthy subjects. BEX fails to explain the observed data and, compared to the new approach, overestimates the speed of emptying. Predictive accuracy under impaired gastric motility was explored using synthetic data. Only the new approach can reproduce a multiphase absorption profile. When routine benchtop equipment was used for measurements, then the rate-limiting step for precision in the estimate of emptying is the quality in the a priori estimate for kinetic parameters rather than precision in measurements. Only about 80% of the absorbed [Formula: see text] has to be released by expiration. With these features, the new approach promises to widen the applicability of breath tests for gastric emptying.

Diminished aqueous microviscosity of tumors in murine models measured with in vivo radiofrequency electron paramagnetic resonance.

Using very low frequency in vivo electron paramagnetic resonance (EPR), we have compared, for the first time, the average microviscosity of the total aqueous compartment of murine fibrosarcomas and that of normal leg tissue in a living animal. EPR spectra from dissolved nitroxide spin probes report the solvent microviscosity. The tumor aqueous microviscosity, 1.8 +/- 0.1 centipoise, was significantly lower than that of the corresponding normal tissue, 2.9 +/- 0.3 centipoise, a difference of 38 +/- 7%. These results confirm the commonly observed increase in the water proton transverse relaxation times (T2) in magnetic resonance imaging of hyperproliferative states, for example, malignancy. The specificity of the localization of the EPR signal indicates a substantial portion of the T2 increase seen in magnetic resonance imaging derives from decreased bulk-water viscosity. The effect of this microviscosity differences may be the basis of several physiological differences between tumors and normal tissues which could confer a growth rate advantage to tumor tissue.

Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies.

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di(N-methyl-D-glucamine-dithiocarbamate) (Fe2+(MGD)2) and the novel chelate ferro-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD)2 and NO-Fe(DTCS)2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO-Fe(MGD)2 and NO-Fe(DTCS)2 in mice in real time.

A geometric framework for nonlinear visual coding.

It is argued that important aspects of early and middle level visual coding may be understood as resulting from basic geometric processing of the visual input. The input is treated as a hypersurface defined by image intensity as a function of two spatial coordinates and time. Analytical results show how the Riemann curvature tensor R of this hypersurface represents speed and direction of motion. Moreover, the results can predict the selectivity of MT neurons for multiple motions and for motion in a direction along the optimal spatial orientation. Finally, a model based on integrated R components predicts global-motion percepts related to the barber-pole illusion.

Psychiatric consultations in stiff-man syndrome.

BACKGROUND: Stiff-man syndrome is a rare central nervous system disease first described nearly 40 years ago. Its cause has been attributed to both neurologic and psychiatric processes. In recent years, it has been accepted as a neurologic condition in which the gamma-aminobutyric acid (GABA) system malfunctions, probably because of an autoimmune process. Published reports that have described psychiatric manifestations of the disease have relied on descriptions of one or two cases and literature reviews. METHOD: We reviewed the medical records of 24 patients with confirmed stiff-man syndrome, 12 of whom had received psychiatric consultation. This review was done to better determine the psychiatric manifestations of stiff-man syndrome. RESULTS: Retrospective analysis of these 12 cases showed that the most common psychiatric symptoms were anxiety, depression, and alcohol abuse. CONCLUSION: We speculate that the GABA system is involved in both the neurologic and psychiatric symptoms of these patients. Psychiatrists have a significant role in the management of patients with stiff-man syndrome and may be expected to manage anxiety, depression, and substance misuse.

Bioactive glass ceramic on titanium substrate: the effect of molybdenum as an intermediate bond coating.

Bioactive glass ceramic coatings have been proposed for cementless prosthetic fixation. However, a coating procedure that results in a bond of adequate strength with the metal substrate is one of several factors that has to be resolved. In this work a glass ceramic coating was plasma flame sprayed onto a titanium substrate with and without molybdenum as an intermediate bond coating. The higher bond strength of the compound (20 MN m-2) compared to the simple (6 MN m-2) coating could be due to an increased roughness offered by the molybdenum surface, and possibly improved chemical bonding to the glass ceramic.

In vitro and in vivo comparative colonization of Staphylococcus aureus and Staphylococcus epidermidis on orthopaedic implant materials.

Clinically, Staphylococcus aureus appears to be the dominant organism associated with infected metal implants, whereas coagulase-negative staphylococcal strains are more frequently isolated from infected polymer implants. We reproduced this trend experimentally in vitro and in vivo. Discs of a titanium alloy, poly(methyl methacrylate) and ultra-high molecular weight polyethylene were exposed to a clinical isolate of Staphylococcus aureus or either of two strains of Staphylococcus epidermidis. Within 1 h Staphylococcus aureus was always the most rapid colonizer regardless of biomaterial. However, after 8 to 24 h, Staphylococcus aureus was present in higher numbers on metal and Staphylococcus epidermidis on polymers. Moreover, the exopolysaccharide produced by Staphylococcus epidermidis appeared to offer an effective protection against host defences in vivo.

Staphylococcal adhesion to collagen in intra-articular sepsis.

Ultrastructural studies of the cartilaginous articular surfaces of human and rabbit joints have shown that cartilage is the target substratum for adhesion by Staphylococcus aureus, leading to intra-articular sepsis. Transmission and scanning electron microscope studies demonstrated bacteria in intimate contact with acellular cartilage matrix surfaces, particularly with collagen fibres. Certain strains of Staphylococcus aureus used in these experiments reveal a high binding capacity to collagen that is derived from a cartilage matrix. These studies indicate that the pathogenesis of intra-articular sepsis is based on the ability of certain strains of staphylococci to bind preferentially to a cartilage matrix.

Measurement of differences in pO2 in response to perfluorocarbon/carbogen in FSa and NFSa murine fibrosarcomas with low-frequency electron paramagnetic resonance oximetry.

We have used very low-frequency electron paramagnetic resonance (EPR) oximetry to measure the change in oxygen concentration (delta pO2) due to change in breathing atmosphere in FSa and NFSa fibrosarcomas implanted in the legs of C3H mice infused with perfluoro-octylbromine (PFOB). Measurements in each tumor were made before and after the administration of the high-density (47% v/v) perfluorocarbon PFOB, perflubron (Alliance Pharmaceutical Corporation, San Diego, CA). Measurements in each tumor were also made, after the administration of the PFOB, both before (PFOB/air) and after the administration of carbogen (95% O2 + 5% CO2, PFOB/carbogen). Large changes (delta p02) relative to PFOB/air oxygenation were seen with the administration of PFOB/carbogen. No significant difference in oxygen concentration was seen between air-breathing mice with and without PFOB. The mean delta pO2 for FSa tumors was 13 +/- 6 torr, while the mean for NFSa fibrosarcomas was 28 +/- 7 torr. There were such large intertumor differences that the trend toward a smaller change in the more hypoxic FSa tumors was not significant (P = 0.13). This paper describes a novel method of measuring differences in oxygenation in tumor tissues. The results of such measurements indicate large differences in pO2 response to different breathing atmospheres in PFOB-infused tumors of similar histology. The intertumor delta pO2 differences may correlate with differences in radiation response.