ABSTRACT
BACKGROUND: Antibiotic treatment is crucial for patients with chronic bacterial infections. Suspected drug allergies often lead to inconsistent therapies and challenging clinical management for patients and caregivers. The objective of this study was to evaluate the value of lymphocyte transformation tests in comparison to skin tests for the prediction of delayed-type allergic reactions. METHODS: This prospective, observational study tested the diagnostic value of skin prick tests, intradermal tests (reading: 15 min and 72 h) and lymphocyte transformations tests for the prediction of allergic reactions in CF patients with physician reported allergy to piperacillin/tazobactam, meropenem and ceftazidime. The tests were performed directly before a 14d intravenous drug challenge. RESULTS: We performed 33 drug challenges in 29 subjects. 21 drug challenges were negative (63 %); 12 lead to a reaction (37 %), of those 2 were immediate and 10 were delayed-type. 100 % of the skin prick tests were negative. 97 % (33/34) of the intradermal tests with early reading and 100 % of the intradermal tests with late reading yielded negative results. 5/11 patients who experienced a delayed-type reaction during the drug challenge had a positive lymphocyte transformations test. All 17 patients who did not react had a negative lymphocyte transformations test. For piperacillin/tazobactam, 4/5 patients who experienced a delayed-type reaction during the drug challenge had positive lymphocyte transformations tests. Hence, for piperacillin/tazobactam, the sensitivity of the lymphocyte transformation test for prediction of reactions was 80.0 % and the specificity 100 %. CONCLUSION: We demonstrate that the lymphocyte transformation test predicts delayed-type allergy to piperacillin/tazobactam in contrast to skin tests.
ABSTRACT
CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Chlorides/therapeutic use , Homozygote , Mutation , Benzodioxoles/pharmacology , Benzodioxoles/therapeutic useABSTRACT
Thermal noise drives cellular structures, bacteria, and viruses on different temporal and spatial scales. Their weak interactions with their environment can change on subsecond scales. However, particle interactions can be hidden or invisible-even when measured with thermal noise sensitivity, leading to misconceptions about their binding behavior. Here, it is demonstrated how invisible particle interactions at the cell periphery become visible by MHz interferometric thermal noise tracking and frequency decomposition at a spectral update rate of only 0.5 s. The particle fluctuations are analyzed in radial and lateral directions by a viscoelastic modulus G(ω,tex ) over the experiment time tex , revealing a surprisingly similar, frequency dependent response for different cell types. This response behavior can be explained by a mathematical model for molecular scale elasticity and damping. The method to reveal hidden interactions is tested at two examples: the stiffening of macrophage filopodia tips within 2 s with particle contact invisible by the fluctuation width. Second, the extent and stiffness of the soft cell glycocalyx is measured, which can be sensed by a particle only on microsecond-timescales, but which remains invisible on time-average. This concept study shows how to uncover hidden cellular interactions, if particle motions are measured at high-speed.
Subject(s)
Macrophages , Models, Theoretical , Cytoplasm , Motion , Macrophages/metabolismABSTRACT
Phagocytic particle uptake is crucial for the fate of both living cells and pathogens. Invading particles have to overcome fluctuating lipid membranes as the first physical barrier. However, the energy and the role of the fluctuation-based particle-membrane interactions during particle uptake are not understood. We tackle this problem by indenting the membrane of differently composed Giant Unilamellar Vesicles (GUVs) with optically trapped particles until particle uptake. By continuous 1 MHz tracking and autocorrelating the particle's positions within 30µs delays for different indentations, the fluctuations' amplitude, the damping, the mean forces, and the energy profiles were obtained. Remarkably, the uptake energy into a GUV becomes predictable since it increases for smaller fluctuation amplitudes and longer relaxation time. Our observations could be explained by a mathematical model based on continuous suppression of fluctuation modes. Hence, the reduced particle uptake energy for protein-ligand interactions LecA-Gb3 or Biotin-Streptavidin results also from pronounced, low-friction membrane fluctuations.
Subject(s)
Models, Theoretical , Unilamellar Liposomes , Biological Transport , Phagocytosis , LipidsABSTRACT
Research in modern light microscopy continuously seeks to improve spatial and temporal resolution in combination with user-friendly, cost-effective imaging systems. Among different label-free imaging approaches, Rotating Coherent Scattering (ROCS) microscopy in darkfield mode achieves superior resolution and contrast without image reconstructions, which is especially helpful in life cell experiments. Here we demonstrate how to achieve 145â nm resolution with an amplitude transmission mask for spatial filtering. This mask blocks the reflected 0-th order focus at 12 distinct positions, thereby increasing the effective aperture for the light back-scattered from the object. We further show how angular correlation analysis between coherent raw images helps to estimate the information content from different illumination directions.
Subject(s)
Image Processing, Computer-Assisted , Microscopy , Microscopy/methods , Image Processing, Computer-Assisted/methods , LightingABSTRACT
Macrophages use filopodia to withdraw particles toward the cell body for phagocytosis. This can require substantial forces, which the cell generates after bio-mechanical stimuli are transmitted to the filopodium. Adaptation mechanisms to mechanical stimuli are essential for cells, but can a cell iteratively improve filopodia pulling? If so, the underlying mechanic adaptation principles organized on the protein level are unclear. Here, we tackle this problem using optically trapped 1 µm beads, which we tracked interferometrically at 1 MHz during connection to the tips of dorsal filopodia of macrophages. We observe repetitive failures while the filopodium tries to pull the bead out of the optical trap. Analyses of mean bead motions and position fluctuations on the nano-meter and microsecond scale indicate mechanical ruptures caused by a force-dependent actin-membrane connection. We found that beads are retracted three times slower under any load between 5 and 40 pN relative to the no-load transport, which has the same speed as the actin retrograde flow obtained from fluorescent speckle tracking. From this duty ratio of pulling velocities, we estimated a continuous on/off binding with τoff = 2â τon, with measured off times τoff = 0.1-0.5 s. Remarkably, we see a gradual increase of filopodia pulling forces from 10 to 30 pN over time and after failures, which points toward an unknown adaptation mechanism. Additionally, we see that the attachment strength and friction between the bead and filopodium tip increases under load and over time. All observations are typical for catch-bond proteins such as integrin-talin complexes. We present a mechanistic picture of adaptive mechanotransduction, which formed by the help of mathematical models for repetitive tip ruptures and reconnections. The analytic mathematical model and the stochastic computer simulations, both based on catch-bond lifetimes, confirmed our measurements. Such catch-bond characteristics could also be important for other immune cells taking up counteracting pathogens.
Subject(s)
Actins , Pseudopodia , Actins/metabolism , Macrophages/metabolism , Mechanotransduction, Cellular , Phagocytosis/physiology , Pseudopodia/metabolismABSTRACT
BACKGROUND: The Dermatophagoides pteronyssinus molecule Der p 23 is a major allergen whose clinical relevance has been shown in cross-sectional studies. We longitudinally analysed the trajectory of Der p 23-specific IgE antibody (sIgE) levels throughout childhood and youth, their early-life determinants and their clinical relevance for allergic rhinitis and asthma. METHODS: We obtained sera and clinical data of 191 participants of the German Multicentre Allergy Study, a prospective birth cohort. Serum samples from birth to 20 years of age with sIgE reactivity to Der p 23 in a customised semiquantitative microarray were newly analysed with a singleplex quantitative assay. Early mite exposure was assessed by measuring the average content of Der p 1 in house dust at 6 and 18 months. RESULTS: Der p 23-sIgE levels were detected at least once in 97/191 participants (51%). Prevalence of Der p 23 sensitisation and mean sIgE levels increased until age 10 years, plateaued until age 13 years and were lowest at age 20 years. Asthma, allergic rhinitis (AR) and atopic dermatitis (AD) were more prevalent in Der p 23-sensitised children, including those with monomolecular but persistent sensitisation (11/97, 11%). A higher exposure to mites in infancy and occurrence of AD before 5 years of age preceded the onset of Der p 23 sensitisation, which in turn preceded a higher incidence of asthma. CONCLUSIONS: Der p 23 sensitisation peaks in late childhood and then decreases. It is preceded by early mite exposure and AD. Asthma and AR can occur in patients persistently sensitised to Der p 23 as the only mite allergen, suggesting the inclusion of molecular testing of Der p 23-sIgE for subjects with clinical suspicion of HDM allergy but without sIgE to other major D.pt. allergens.
Subject(s)
Asthma , Dermatitis, Atopic , Mites , Rhinitis, Allergic , Adolescent , Adult , Allergens , Animals , Antigens, Dermatophagoides , Birth Cohort , Child , Cohort Studies , Cross-Sectional Studies , Humans , Immunoglobulin E , Prospective Studies , Young AdultABSTRACT
Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions.
Subject(s)
Actins , Lighting , Fibroblasts , Microscopy, Fluorescence/methodsABSTRACT
Rationale: The CFTR (cystic fibrosis transmembrane conductance regulator) modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) was shown to improve clinical outcomes and sweat chloride concentration in patients with cystic fibrosis (CF) and one or two F508del alleles. However, the effect of ELX/TEZ/IVA on CFTR function in the airways and intestine has not been studied. Objectives: To assess the effect of ELX/TEZ/IVA on CFTR function in airway and intestinal epithelia in patients with CF and one or two F508del alleles aged 12 years and older. Methods: This prospective, observational, multicenter study assessed clinical outcomes including FEV1% predicted and body mass index and the CFTR biomarkers sweat chloride concentration, nasal potential difference, and intestinal current measurement before and 8-16 weeks after initiation of ELX/TEZ/IVA. Measurements and Main Results: A total of 107 patients with CF including 55 patients with one F508del and a minimal function mutation and 52 F508del homozygous patients were enrolled in this study. In patients with one F508del allele, nasal potential difference and intestinal current measurement showed that ELX/TEZ/IVA improved CFTR function in nasal epithelia to a level of 46.5% (interquartile range [IQR], 27.5-72.4; P < 0.001) and in intestinal epithelia to 41.8% of normal (IQR, 25.1-57.6; P < 0.001). In F508del homozygous patients, ELX/TEZ/IVA exceeded improvement of CFTR function observed with TEZ/IVA and increased CFTR-mediated Cl- secretion to a level of 47.4% of normal (IQR, 19.3-69.2; P < 0.001) in nasal and 45.9% (IQR, 19.7-66.6; P < 0.001) in intestinal epithelia. Conclusions: Treatment with ELX/TEZ/IVA results in effective improvement of CFTR function in airway and intestinal epithelia in patients with CF and one or two F508del alleles. Clinical trial registered with www.clinicaltrials.gov (NCT04732910).
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Alleles , Aminophenols/therapeutic use , Benzodioxoles/therapeutic use , Chloride Channel Agonists/therapeutic use , Chlorides , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Humans , Indoles , Mutation , Prospective Studies , Pyrazoles , Pyridines , Pyrrolidines , QuinolonesABSTRACT
In modern 3D microscopy, holding and orienting arbitrary biological objects with optical forces instead of using coverslips and gel cylinders is still a vision. Although optical trapping forces are strong enough and related photodamage is acceptable, the precise (re-) orientation of large specimen with multiple optical traps is difficult, since they grab blindly at the object and often slip off. Here, we present an approach to localize and track regions with increased refractive index using several holographic optical traps with a single camera in an off-focus position. We estimate the 3D grabbing positions around several trapping foci in parallel through analysis of the beam deformations, which are continuously measured by defocused camera images of cellular structures inside cell clusters. Although non-blind optical trapping is still a vision, this is an important step towards fully computer-controlled orientation and feature-optimized laser scanning of sub-mm sized biological specimen for future 3D light microscopy.
Subject(s)
Interferometry/methods , Microscopy/methods , Refractometry/methods , Cell Line, Tumor , Humans , Interferometry/instrumentation , Microscopy/instrumentation , Models, Theoretical , Optical Tweezers , Refractometry/instrumentationABSTRACT
Rotating coherent scattering (ROCS) microscopy is a label-free imaging technique that overcomes the optical diffraction limit by adding up the scattered laser light from a sample obliquely illuminated from different angles. Although ROCS imaging achieves 150 nm spatial and 10 ms temporal resolution, simply summing different speckle patterns may cause loss of sample information. In this paper we present Deep-ROCS, a neural network-based technique that generates a superior-resolved image by efficient numerical combination of a set of differently illuminated images. We show that Deep-ROCS can reconstruct super-resolved images more accurately than conventional ROCS microscopy, retrieving high-frequency information from a small number (6) of speckle images. We demonstrate the performance of Deep-ROCS experimentally on 200 nm beads and by computer simulations, where we show its potential for even more complex structures such as a filament network.
ABSTRACT
Interactions of the bacterial lectin LecA with the host cells glycosphingolipid Gb3 have been shown to be crucial for the cellular uptake of the bacterium Pseudomonas aeruginosa. LecA-induced Gb3 clustering, referred to as lipid zipper mechanism, leads to full membrane engulfment of the bacterium. Here, we aim for a nanoscale force characterization of this mechanism using two complementary force probing techniques, atomic force microscopy (AFM) and optical tweezers (OT). The LecA-Gb3 interactions are reconstituted using giant unilamellar vesicles (GUVs), a well-controlled minimal system mimicking the plasma membrane and nanoscale forces between either bacteria (PAO1 wild-type and LecA-deletion mutant strains) or LecA-coated probes (as minimal, synthetic bacterial model) and vesicles are measured. LecA-Gb3 interactions strengthen the bacterial attachment to the membrane (1.5-8-fold) depending on the membrane tension and the applied technique. Moreover, significantly less energy (reduction up to 80%) is required for the full uptake of LecA-coated beads into Gb3-functionalized vesicles. This quantitative approach highlights that lectin-glycolipid interactions provide adequate forces and energies to drive bacterial attachment and uptake.
Subject(s)
Adhesins, Bacterial , Lectins , Adhesins, Bacterial/metabolism , Biological Transport , Cell Membrane/metabolism , Lectins/metabolism , Pseudomonas aeruginosa/metabolism , Unilamellar Liposomes/metabolismSubject(s)
Aminopyridines/adverse effects , Benzodioxoles/adverse effects , CD4-Positive T-Lymphocytes/immunology , Cystic Fibrosis/drug therapy , Drug Hypersensitivity/immunology , Aminophenols/therapeutic use , Aminopyridines/therapeutic use , Benzodioxoles/therapeutic use , Cystic Fibrosis Transmembrane Conductance Regulator , Drug Combinations , Drug Hypersensitivity/etiology , Female , Humans , Quinolones/therapeutic use , Young AdultABSTRACT
Fluorescence microscopy is the standard imaging technique to investigate the structures and dynamics of living cells. However, increasing the spatial resolution comes at the cost of temporal resolution and vice versa. In addition, the number of images that can be taken in sufficiently high quality is limited by fluorescence bleaching. Hence, super-resolved imaging at several Hertz of low fluorescent biological samples is still a big challenge and, especially in structured illumination microscopy (SIM), is often visible as imaging artifacts. In this paper, we present a TIRF-SIM system based on scan-mirrors and a Michelson interferometer, which generates images at 110 nm spatial resolution and up to 8 Hz temporal resolution. High resolution becomes possible by optimizing the illumination interference contrast, even for low fluorescent, moving samples. We provide a framework and guidelines on how the modulation contrast, which depends on laser coherence, polarization, beam displacement or sample movements, can be mapped over the entire field of view. In addition, we characterize the influence of the signal-to-noise ratio and the Wiener filtering on the quality of reconstructed SIM images, both in real and frequency space. Our results are supported by theoretical descriptions containing the parameters leading to image artifacts. This study aims to help microscopists to better understand and adjust optical parameters for structured illumination, thereby leading to more trustworthy measurements and analyses of biological dynamics.
ABSTRACT
The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis.
ABSTRACT
BACKGROUND: House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. OBJECTIVE: Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. METHODS: Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. RESULTS: Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. CONCLUSION: Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.
Subject(s)
Antigens, Dermatophagoides/immunology , Arthropod Proteins/immunology , Cysteine Endopeptidases/immunology , Desensitization, Immunologic/methods , Hypersensitivity/therapy , Immunoglobulin E/metabolism , Immunoglobulin G/metabolism , Plant Extracts/therapeutic use , Adult , Animals , Epitopes/immunology , Female , Humans , Hypersensitivity/immunology , Injections, Subcutaneous , Male , Protein Array Analysis , PyroglyphidaeABSTRACT
Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here, we show that it is possible to detect single binding events of immunoglobulin-G-coated polystyrene beads, which are held in an optical trap near the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically, and no fluorophore labeling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential stepwise increases in the force constant of the bond between a bead and a cell of typically 20 pN/µm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes, including phagocytosis, drug delivery, and the effects of small microplastics and particulates on cells.
Subject(s)
Plastics , Biophysical Phenomena , Cell Membrane , Diffusion , MotionABSTRACT
PURPOSE: Wheat is a frequent elicitor of food allergy in childhood. Especially in popular media, the better digestibility and the lower allergenicity of ancient grains are repeatedly postulated. We addressed the question whether ancient wheat-related grains are less allergenic than modern wheat. METHODS: Proteins from cultivars of spelt, einkorn, emmer and durum along with durum soft wheat flour, Tritordeum and bread wheat were separated by electrophoresis. Immunoblots were performed with a pool serum of six sera from wheat-sensitized children aged 1-11 years (wheat-specific IgE 22 kUA/l). As controls, pool serum from five sera atopic patients aged 3-13 years who had no sensitization to wheat (wheat-specific IgE 0.11 kUA/l) and six sera from non-atopics at the age of 3 months to 5 years (wheat-specific IgE 0.06 kUA/l) was used. Area under the curve (AUC) in Coomassie-stained gels and immunoblots was determined and related. RESULTS: Water/salt-soluble protein patterns were very similar among varieties. In einkorn cultivars, one protein band corresponding to an alpha-amylase/trypsin inhibitor (ATI) was absent. Water-insoluble protein fractions differed markedly among varieties and cultivars. IgE bound to a large number of proteins in all protein fractions both in wheat and in the wheat-related ancient grains. CONCLUSIONS: Patients with sensitization to wheat show a significant IgE binding against both modern and ancient grain varieties of the genus Triticum. Therefore, ancient grains do not appear to have a generally reduced risk for wheat allergy sufferers. However, few individuals only sensitized to ATI could benefit from the consumption of einkorn.
Subject(s)
Food Hypersensitivity , Wheat Hypersensitivity , Allergens , Child , Flour , Humans , TriticumABSTRACT
BACKGROUND: There is growing interest both in testing IgE in nasal secretions (NS) and in molecular diagnosis of seasonal allergic rhinitis (SAR). Yet, the reliability of nasal IgE detection with the newest molecular assays has never been assessed in a large cohort of pollen allergic patients. OBJECTIVE: To investigate with microarray technology and compare the repertoires of specific IgE (sIgE) antibodies in NS and sera of a large population of children and adults with SAR. METHODS: Nasal secretions were collected with an absorbent device (Merocel 2000® , Medtronic) and a minimal dilution procedure from 90 children and 71 adults with SAR. Total IgE (tIgE) (ImmunoCAP, Thermo Fisher Scientific (TFS)) and sIgE antibodies against 112 allergen molecules (ISAC-112, TFS) were measured in NS and serum. RESULTS: Nasal sIgE was detectable in 68.3% of the patients. The detected nasal sIgE antibodies recognized airborne (88%), vegetable (10%), and animal food or other (<1%) allergen molecules. The prevalence and average levels of sIgE in NS and serum were highly interrelated at population level. A positive nasal sIgE antibody to a given molecule predicted the detection of the same antibody in the patient's serum with a specificity of 99.7% and a sensitivity of 40%. CONCLUSIONS: The concentration of sIgE is much lower in nasal secretions than in the serum. sIgE assays with very high analytical sensitivity and sampling methods with minimal dilution will be therefore needed to validate nasal secretions as alternative to serum in testing the sIgE repertoire.
Subject(s)
Bodily Secretions/immunology , Immunoglobulin E/isolation & purification , Nose/immunology , Rhinitis, Allergic, Seasonal/diagnosis , Rhinitis, Allergic, Seasonal/immunology , Adolescent , Adult , Aged , Allergens/immunology , Animals , Child , Cohort Studies , Humans , Immunoglobulin E/blood , Microarray Analysis , Middle Aged , Pollen/immunology , Rhinitis, Allergic, Seasonal/blood , Vegetables/immunology , Young AdultABSTRACT
In light-sheet microscopy, a confined layer in the focal plane of the detection objective is illuminated from the side. The illumination light-sheet usually has a constant beam length independent of the shape of the biological object. Since the thickness and the length of the illumination light-sheet are coupled, a tradeoff between resolution, contrast and field of view has to be accepted. Here we show that scanned Bessel beams enable object adapted tailoring of the light-sheet defined by its beam length and position. The individual beam parameters are obtained from automatic object shape estimation by low-power laser light scattered at the object. Using Arabidopsis root tips, cell clusters and zebrafish tails, we demonstrate that Bessel beam light-sheet tailoring leads to a 50% increase in image contrast and a 50% reduction in photobleaching. Light-sheet tailoring requires only binary amplitude modulation, therefore allowing a real time illumination adaptation with little technical effort in the future.