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Low antigen sensitivity and a gradual loss of effector functions limit the clinical applicability of chimeric antigen receptor (CAR)-modified T cells and call for alternative antigen receptor designs for effective T cell-based cancer immunotherapy. Here, we applied advanced microscopy to demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform second-generation CAR formats with regard to conveyed antigen sensitivities by up to a thousandfold. TCC-based antigen recognition occurred without adverse nonspecific signaling, which is typically observed in CAR-T cells, and did not depend-unlike sensitized peptide/MHC detection by conventional T cells-on CD4 or CD8 coreceptor engagement. TCC-endowed signaling properties may prove critical when targeting antigens in low abundance and aiming for a durable anticancer response.

Receptor-ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR-pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists. Traditional surface plasmon resonance (SPR) methods quantify 3D affinity but lack cellular context and fail to account for factors like membrane fluctuations. In the recent years, single-molecule Förster resonance energy transfer (smFRET) has been applied to measure 2D binding kinetics of TCR-pMHC interactions in a cellular context. Here, we introduce a rigorous mathematical model based on survival analysis to determine exponentially distributed receptor-ligand interaction lifetimes, verified through simulated data. Additionally, we developed a comprehensive analysis pipeline to extract interaction lifetimes from raw microscopy images, demonstrating the model's accuracy and robustness across multiple TCR-pMHC pairs. Our new software suite automates data processing to enhance throughput and reduce bias. This methodology provides a refined tool for investigating T cell activation mechanisms, offering insights into immune response modulation.

Patients with multiple myeloma (MM) are a heterogenous, immunocompromised group with increased risk for COVID-19 morbidity and mortality but impaired responses to primary mRNA SARS-CoV-2 vaccination. The effects of booster vaccinations and breakthrough infections (BTIs) on antibody (Ab) levels and cross-protection to variants of concern (VOCs) are, however, not sufficiently evaluated. Therefore, we analysed humoral and cellular vaccine responses in MM patients stratified according to disease stage/treatment into group (1) monoclonal gammopathy of undetermined significance, (2) after stem cell transplant (SCT) without immunotherapy (IT), (3) after SCT with IT, and (4) progressed MM, and in healthy subjects (prospective cohort study). In contrast to SARS-CoV-2 hu-1-specific Ab levels, Omicron-specific Abs and their cross-neutralisation capacity remained low even after three booster doses in a majority of MM patients. In particular, progressed MM patients receiving anti-CD38 mAb and those after SCT with IT were Ab low responders and showed delayed formation of spike-specific B memory cells. However, MM patients with hybrid immunity (i.e., vaccination and breakthrough infection) had improved cross-neutralisation capacity against VOCs, yet in the absence of severe COVID-19 disease. Our results indicate that MM patients require frequent variant-adapted booster vaccinations and/or changes to other vaccine formulations/platforms, which might have similar immunological effects as BTIs.

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The humanized monoclonal anti-α4β7-integrin-antibody vedolizumab is one of several biologic therapeutic options in moderate-to-severe ulcerative colitis and Crohn's disease. Within the VISIBLE trial program, a novel subcutaneous application route was evaluated in addition to the already established intravenous form. In this position statement, the working group "Inflammatory Bowel Diseases" of the Austrian Society for Gastroenterology and Hepatology (OEGGH) summarizes the evidence regarding the subcutaneous application of vedolizumab. This work supplements a position paper on the value of vedolizumab as a first-line biologic that has already been published and offers useful recommendations for clinical practice., Competing Interests: Die Autoren erklären, dass die Erstellung dieses Positionspapieres finanziell von TAKEDA Austria unterstützt wurde und dass der Erstautor (S.R.) innerhalb der letzten 3 Jahre Vortragshonorare von TAKEDA Austria erhalten hat., (Thieme. All rights reserved.)

Background: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown., Methods: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models., Results: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11 -knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11 -knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42., Conclusions: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.)., (Copyright © 2023 Massachusetts Medical Society.)

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4-LCK and CD8-LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8 + T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor-LCK interactions as promising targets for immunomodulation., (© 2022. The Author(s).)

When T cells scan the surface of antigen-presenting cells (APCs), they can detect the presence of just a few antigenic peptide/MHC complexes (pMHCs), in some cases even a single agonist pMHC. These are typically vastly outnumbered by structurally similar yet non-stimulatory endogenous pMHCs. How T cells achieve this enormous sensitivity and selectivity is still not clear, in particular in view of the rather moderate (1-100 μM) affinity that T-cell receptors (TCRs) typically exert for antigenic pMHCs. Experimental approaches that enable the control and quantification of physical input parameters within the context of the immunological synapse to precisely interrogate the molecular consequences of TCR-engagement, appear highly advantageous when searching for better answers.We here describe the implementation of a biointerface that allows to experimentally define molecular distances between T-cell ligands as a means to correlate them with molecular dynamics of antigen engagement, downstream signaling, and the overall T-cell response. The basis of this biointerface is DNA origami nanostructures, which are (i) rigid and highly versatile platforms that can (ii) be embedded as laterally mobile entities within supported lipid bilayers and functionalized (iii) in a site-specific and orthogonal manner with (iv) one or more proteins of choice., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry, and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as the protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to three proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral peptide nucleic acid (PNA) in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

CD4 + T cell trafficking is a fundamental property of adaptive immunity. In this study, we uncover a novel role for histone deacetylase 1 (HDAC1) in controlling effector CD4 + T cell migration, thereby providing mechanistic insight into why a T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis (EAE). HDAC1-deficient CD4 + T cells downregulated genes associated with leukocyte extravasation. In vitro, HDAC1-deficient CD4 + T cells displayed aberrant morphology and migration on surfaces coated with integrin LFA-1 ligand ICAM-1 and showed an impaired ability to arrest on and to migrate across a monolayer of primary mouse brain microvascular endothelial cells under physiological flow. Moreover, HDAC1 deficiency reduced homing of CD4 + T cells into the intestinal epithelium and lamina propria preventing weight-loss, crypt damage and intestinal inflammation in adoptive CD4 + T cell transfer colitis. This correlated with reduced expression levels of LFA-1 integrin chains CD11a and CD18 as well as of selectin ligands CD43, CD44 and CD162 on transferred circulating HDAC1-deficient CD4 + T cells. Our data reveal that HDAC1 controls T cell-mediated autoimmunity via the regulation of CD4 + T cell trafficking into the CNS and intestinal tissues., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Patients with inflammatory bowel disease (IBD) suffer from various symptoms, impairing their quality of life and often affecting psychosocial issues. This may lead to the need for additional psychological care. This study investigated patients' subjective need for integrated psychosomatic support and psychotherapy and indicators for it., Materials and Methods: This is a cross-sectional multicentre study in Austrian IBD patients who were in routine care at 18 IBD outpatient clinics. Patients filled in an anonymous, validated questionnaire (Assessment of the Demand for Additional Psychological Treatment Questionnaire [ADAPT]) assessing the need for psychological care. The ADAPT gives two separate scores: the need for integrated psychosomatic support and for psychotherapy. In addition, health-related quality of life and the use of complementary and alternative medicine as well as clinical and socio-demographic variables were queried. Multivariable regression analysis was performed to estimate the effect of the previously mentioned variables on the need for additional psychological care., Results: Of 1286 patients, 29.7% expressed a need for additional psychological care, 19.6% expressed a need for integrated psychosomatic support and 20.2% expressed a need for psychotherapy. In the multivariable analysis, the two strongest indicators for the need for both types of psychological care were the use of complementary and alternative medicine (for integrated psychosomatic support: odds ratio = 1.64, 95% confidence interval 1.13-2.39, p = 0.010; for psychotherapy: odds ratio = 1.74, 95% confidence interval 1.20-2.53, p = 0.004), and a low health-related quality of life score (for integrated psychosomatic support: odds ratio = 0.95, 95% confidence interval 0.94-0.96, p < 0.001; for psychotherapy: odds ratio = 0.96, 95% confidence interval 0.94-0.97, p < 0.001)., Discussion: About 30% of the Austrian IBD patients expressed a need for integrated psychosomatic support and/or psychotherapy. The most important indicators for this need were the use of complementary and alternative medicine and low quality of life., (© 2020 The Authors. United European Gastroenterology Journal published by Wiley Periodicals LLC. on behalf of United European Gastroenterology.)

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules., Competing Interests: The authors declare no competing interest.

T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction of costimulatory and adhesion proteins, remodeling of the actin cytoskeleton, and the initiation of downstream signaling processes such as the release of intracellular calcium. However, multiparametric time-resolved analysis of these processes is hampered by the difficulty in recording the different readout modalities at high quality in parallel. In this study, we present a platform for simultaneous quantification of TCR distribution via total internal reflection fluorescence microscopy, of intracellular calcium levels, and of T-cell-exerted forces via atomic force microscopy (AFM). In our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. We designed the platform specifically to enable the study of T-cell triggering via functionalized fluid-supported lipid bilayers, which represent a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells triggered specifically via their cognate antigen presented by MHCII.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin, and failure to assemble lamellipodia. Hem1 -/- mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B cell fate choices toward autoreactive B cells and thus autoimmunity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Objective: Complementary and alternative medicine (CAM) seems to be frequently used among patients with inflammatory bowel disease (IBD). We aimed to determine the prevalence and indicators of CAM use in Austrian IBD patients., Methods: In a multicentre cross-sectional study, adult patients with IBD attending 18 Austrian outpatient clinics completed a multi-item questionnaire that recorded use of CAM as well as medical and socioeconomic characteristics. Patients were recruited between June 2014 and June 2015. The study outcome was the prevalence of CAM use and its socioeconomic and disease-related associations., Results: A total of 1286 patients (Crohn's disease 830, ulcerative colitis 435, IBD unclassified 21; females 651) with a median age of 40 years (interquartile range 31-52 years) and a median disease duration of 10 years (4-18 years) were analysed. The prevalence of previous and/or current CAM use was 50.7%, with similar results for Crohn's disease and ulcerative colitis. In the multivariable analysis, female gender and a university education were independent socioeconomic indicators of CAM use. IBD-related indicators were longer duration of the disease and previous and/or current treatment with steroids and TNF-α inhibitors., Conclusion: CAM use for IBD is frequent in Austrian IBD patients and associated with female gender, higher educational level of university degree, longer duration of the disease, and treatment with steroids and TNF-α inhibitors.

Background: Delayed diagnosis seems to be common in inflammatory bowel diseases (IBD). The study was carried out to investigate the diagnostic delay and associated risk factors in Austrian IBD patients., Methods: In a multicenter cross-sectional study adult patients with IBD attending 18 Austrian outpatient clinics completed a multi-item questionnaire that recorded medical and socioeconomic characteristics. The study outcome was diagnostic delay defined as the period from symptom onset to diagnosis of IBD., Results: A total of 1286 patients (Crohn's disease 830, ulcerative colitis 435, inflammatory bowel disease unclassified 21; females 651) with a median age of 40 years (interquartile range 31-52 years) and a median disease duration of 10 years (4-18 years) were analyzed. The median diagnostic delay was 6 months (2-23 months) in Crohn's disease and 3 months (1-10 months) in ulcerative colitis (p < 0.001). In the multivariable regression analysis Crohn's disease, greater age at diagnosis and a high educational level (compared to middle degree level) were independently associated with longer diagnostic delay., Conclusion: The diagnostic delay was longer in Crohn's disease than in ulcerative colitis patients and was associated with greater age at diagnosis and a higher educational level.

The main function of T cells is to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data have indicated that global clustering of T cell antigen receptors (TCRs) occurs before T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine tunes the T cell response. We found here that apparent TCR nanoclustering could be attributed to overcounting artifacts inherent to single-molecule-localization microscopy. Using complementary super-resolution approaches and statistical image analysis, we found no indication of global nanoclustering of TCRs on antigen-experienced CD4 + T cells under non-activating conditions. We also used extensive simulations of super-resolution images to provide quantitative limits for the degree of randomness of the TCR distribution. Together our results suggest that the distribution of TCRs on the plasma membrane is optimized for fast recognition of antigen in the first phase of T cell activation.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ + CD39 + CD73 + Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

In this paper a numerical technique is presented to compensate for anisotropic optical aberrations, which are usually present across the lateral field of view in the out of focus regions, in high resolution optical coherence tomography and microscopy (OCT/OCM) setups. The recorded enface image field at different depths in the tomogram is digitally divided into smaller sub-regions or the regions of interest (ROIs), processed individually using subaperture based digital adaptive optics (DAO), and finally stitched together to yield a final image with a uniform diffraction limited resolution across the entire field of view (FOV). Using this method, a sub-micron lateral resolution is achieved over a depth range of 218 [Formula: see text]for a nano-particle phantom sample imaged using a fiber based point scanning spectral domain (SD) OCM system with a limited depth of focus (DOF) of ~7 [Formula: see text]at a numerical aperture (NA) of 0.6. Thus, an increase in DOF by ~30x is demonstrated in this case. The application of this method is also shown in ex vivo mouse adipose tissue.

The increment of serum TSH after thyrotrophin-releasing hormone (TRH) is usually considered an extremely sensitive thyroid test, particularly useful in borderline disturbances. We evaluated 195 patients by measuring serum TSH before and 3 h after 40 mg TRH po, and by various other thyroid tests. We found a very tight correlation between the TSH- increment and the 3 h TSH. Thus the TSH-increment can reliably be estimated from the 3 h TSH. The mann-Whitney test confirmed that the 3 h TSH identified thyroid dysfunction as well as the TSH-increment. The basal TSH can therefore be omitted and we propose the measurement of the 3 h TSH as an initial screening test. This single measurement economically excludes the many euthyroid patients sent for thyroid evaluation from further costly tests.

The kinetics of isosorbide dinitrate (ISDN) after i.v. administration and the absolute availability of an oral slow release preparation (SR) were studied in young healthy volunteers. ISDN and the 2- and 5-mononitrates of isosorbide (2-MN, 5-MN) were determined by GLC. After i.v. administration plasma levels of ISDN declined biexponentially and could be adequately described by an open two compartment body model. Distribution half-life was extremely rapid (2-5 min). Terminal disappearance had a half-life of 67 (62-75) min (mean, range). Total plasma clearance was 1.6 (1.2-2.2) litres X min-1, thus approaching liver blood flow. Nevertheless, absolute systemic availability (F) or oral ISDN amounted to 22% (16-29%). Assuming that oral ISDN is completely absorbed and blood levels do not exceed serum levels, an upper limit of hepatic clearance (liver blood flow 1.5 litres X min-1 X (1-F/100)) can be estimated, which is significantly smaller (p less than 0.05) than the measured clearance. This finding is best interpreted by assuming that ISDN is partly eliminated by extrahepatic routes, which is further substantiated by a different pattern of metabolites after i.v. and oral dosing. Whereas after i.v. administration more 2-MN is produced, 5-MN is the main metabolite after oral ISDN. Since the glutathione-S-transferases are found in the cytosol of most cells, it seems likely that other organs than the liver contribute to the metabolism of ISDN.

Isosorbide dinitrate (ISDN) was given to eight healthy volunteers either as 5 mg chewable ISDN hourly or as 40-mg, slow-release formulation every 8 h. Plasma concentration of ISDN and its two metabolites were measured during one dosing interval at steady state. Mean steady-state plasma concentration (Cpss) during the interval was taken as a measure for bioavailability. Cpss after bucal absorption was similar to that after the sustained-release preparation. Relative bioavailability of the latter reached 111%. The effect of ISDN was measured by digital plethysmography (DPG) during the first dosing interval and after 10 and 48 h, when a steady state had been reached with each formulation. DPG changes were of similar magnitude during the first 10 h, but showed a 30% decrease after 48 h. This tolerance is probably due to physiologic counterregulation. Changes in DPG reflect effects of ISDN which can be used during short-term application of the drug to assess systemic bioavailability. Development of tolerance, however, precludes the use of DPG to assess bioavailability in long-lasting steady-state experiments.