Lymphocyte transformation tests predict delayed-type allergy to piperacillin/tazobactam in patients with cystic fibrosis.

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.

Personalized CFTR Modulator Therapy for and Homozygous Patients with Cystic Fibrosis.

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 mutations responsive in Fischer rat thyroid cells, including , but not . However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited.

Making Hidden Cell Particle Interactions Visible by Thermal Noise Frequency Decomposition.

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.

Thermal fluctuations of the lipid membrane determine particle uptake into Giant Unilamellar Vesicles.

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.

ROCS microscopy with distinct zero-order blocking.

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 145 nm resolution with an amplitude transmission mask for spatial filtering.

Pulling, failing, and adaptive mechanotransduction of macrophage filopodia.

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.

Der p 23-specific IgE response throughout childhood and its association with allergic disease: A birth cohort study.

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.

100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales.

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.

Effects of Elexacaftor/Tezacaftor/Ivacaftor Therapy on CFTR Function in Patients with Cystic Fibrosis and One or Two Alleles.

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 alleles. However, the effect of ELX/TEZ/IVA on CFTR function in the airways and intestine has not been studied. To assess the effect of ELX/TEZ/IVA on CFTR function in airway and intestinal epithelia in patients with CF and one or two alleles aged 12 years and older.

Towards non-blind optical tweezing by finding 3D refractive index changes through off-focus interferometric tracking.

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.

Deep-ROCS: from speckle patterns to superior-resolved images by deep learning in rotating coherent scattering microscopy.

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.

Quantification of nanoscale forces in lectin-mediated bacterial attachment and uptake into giant liposomes.

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).

Fast TIRF-SIM imaging of dynamic, low-fluorescent biological samples.

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.

Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes.

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.

Molecular profiling of allergen-specific antibody responses may enhance success of specific immunotherapy.

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.

Measuring Stepwise Binding of Thermally Fluctuating Particles to Cell Membranes without Fluorescence.

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.

IgE antibody repertoire in nasal secretions of children and adults with seasonal allergic rhinitis: A molecular analysis.

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.

Wheat-induced food allergy in childhood: ancient grains seem no way out.

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.

Light-sheet microscopy with length-adaptive Bessel beams.

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.

Miniature scanning light-sheet illumination implemented in a conventional microscope.

Living cells are highly dynamic systems responding to a large variety of biochemical and mechanical stimuli over minutes, which are well controlled by e.g. optical tweezers.

Strong cytoskeleton activity on millisecond timescales upon particle binding revealed by ROCS microscopy.

Cells change their shape within seconds, cellular protrusions even on subsecond timescales enabling various responses to stimuli of approaching bacteria, viruses or pharmaceutical drugs. Typical response patterns are governed by a complex reorganization of the actin cortex, where single filaments and molecules act on even faster timescales. These dynamics have remained mostly invisible due to a superposition of slow and fast motions, but also due to a lack of adequate imaging technology.

Dynamics of a Protein Chain Motor Driving Helical Bacteria under Stress.

The wall-less, helical bacterial genus Spiroplasma has a unique propulsion system; it is not driven by propeller-like flagella but by a membrane-bound, cytoplasmic, linear motor that consists of a contractile chain of identical proteins spanning the entire cell length. By a coordinated spread of conformational changes of the proteins, kinks propagate in pairs along the cell body. However, the mechanisms for the initiation or delay of kinks and their coordinated spread remain unclear.

Label-free Imaging and Bending Analysis of Microtubules by ROCS Microscopy and Optical Trapping.

Mechanical manipulation of single cytoskeleton filaments and their monitoring over long times is difficult because of fluorescence bleaching or phototoxic protein degradation. The integration of label-free microscopy techniques, capable of imaging freely diffusing, weak scatterers such as microtubules (MTs) in real-time, and independent of their orientation, with optical trapping and tracking systems, would allow many new applications. Here, we show that rotating-coherent-scattering microscopy (ROCS) in dark-field mode can also provide strong contrast for structures far from the coverslip such as arrangements of isolated MTs and networks.

Single microtubules and small networks become significantly stiffer on short time-scales upon mechanical stimulation.

The transfer of mechanical signals through cells is a complex phenomenon. To uncover a new mechanotransduction pathway, we study the frequency-dependent transport of mechanical stimuli by single microtubules and small networks in a bottom-up approach using optically trapped beads as anchor points. We interconnected microtubules to linear and triangular geometries to perform micro-rheology by defined oscillations of the beads relative to each other.