| 1. |
- Simoes Cunha, Mafalda, et al.
(author)
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Symptoms of anxiety and depression in patients with persistent asthma : a cross-sectional analysis of the INSPIRERS studies
- 2023
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In: BMJ Open. - : BMJ Publishing Group Ltd. - 2044-6055. ; 13:5
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Journal article (peer-reviewed)abstract
- ObjectivesAnxiety and depression are relevant comorbidities in asthma, but, in Portugal and Spain, data on this topic are scarce. We assessed, in patients with asthma, the frequency of anxiety and depression using the Hospital Anxiety and Depression Scale (HADS) and the European Quality of Life Five Dimension Questionnaire (EQ-5D); the level of agreement between these questionnaires, and the factors associated with these symptoms.MethodsThis is a secondary analysis of the INSPIRERS studies. A total of 614 adolescents and adults with persistent asthma (32.6 +/- 16.9 years, 64.7% female) were recruited from 30 primary care centres and 32 allergy, pulmonology and paediatric clinics. Demographic and clinical characteristics, HADS and EQ-5D were collected. A score >= 8 on Hospital Anxiety and Depression Scale-Anxiety/Hospital Anxiety and Depression Scale-Depression or a positive answer to EQ-5D item 5 indicated the presence of these symptoms. Agreement was determined by Cohen's kappa. Two multivariable logistic regressions were built.ResultsAccording to HADS, 36% of the participants had symptoms of anxiety and 12% of depression. According to EQ-5D, 36% of the participants had anxiety/depression. The agreement between questionnaires in identifying anxiety/depression was moderate (k=0.55, 95% CI 0.48 to 0.62). Late asthma diagnosis, comorbidities and female gender were predictors of anxiety/depression, while better asthma control, health-related quality of life and perception of health were associated with lower odds for anxiety/depression.ConclusionAt least 1/3 of the patients with persistent asthma experience symptoms of anxiety/depression, showing the relevance of screening these disorders in patients with asthma. EQ-5D and HADS questionnaires showed a moderate agreement in the identification of anxiety/depression symptoms. The identified associated factors need to be further investigated in long-term studies.
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| 2. |
- Chouliara, Manto, et al.
(author)
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Single-cell measurements of two-dimensional binding affinity across cell contacts
- 2021
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495. ; 120:22, s. 5032-5040
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Journal article (peer-reviewed)abstract
- The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/μm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/μm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.
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| 3. |
- Fernandes, Ricardo A., et al.
(author)
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A cell topography-based mechanism for ligand discrimination by the T cell receptor
- 2019
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In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:28, s. 14002-14010
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Journal article (peer-reviewed)abstract
- The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.
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| 4. |
- Junghans, Victoria, et al.
(author)
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Dimensions and Interactions of Large T-Cell Surface Proteins
- 2018
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9
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Research review (peer-reviewed)abstract
- The first step of the adaptive immune response involves the interaction of T cells that express T-cell receptors (TCRs) with peptide-loaded major histocompatibility complexes expressed by antigen-presenting cells (APCs). Exactly how this leads to activation of the TCR and to downstream signaling is uncertain, however. Recent findings suggest that one of the key events is the exclusion of the large receptor-type tyrosine phosphatase CD45, from close contacts formed at sites of T-cell/APC interaction. If this is true, a full understanding of how close contact formation leads to signaling would require insights into the structures of, and interactions between, large membrane proteins like CD45 and other proteins forming the glycocalyx, such as CD43. Structural insights into the overall dimensions of these proteins using crystallographic methods are hard to obtain, and their conformations on the cell surface are also unknown. Several imaging-based optical microscopy techniques have however been developed for analyzing protein dimensions and orientation on model cell surfaces with nanometer precision. Here we review some of these methods with a focus on the use of hydrodynamic trapping, which relies on liquid flow from a micropipette to move and trap membrane-associated fluorescently labeled molecules. Important insights that have been obtained include (i) how protein flexibility and coverage might affect the effective heights of these molecules, (ii) the height of proteins on the membrane as a key parameter determining how they will distribute in cell-cell contacts, and (iii) how repulsive interactions between the extracellular parts of the proteins influences protein aggregation and distribution.
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| 5. |
- Junghans, Victoria, et al.
(author)
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Effects of a local auxiliary protein on the two-dimensional affinity of a TCR-peptide MHC interaction
- 2020
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In: Journal of Cell Science. - : The Company of Biologists Ltd.. - 0021-9533 .- 1477-9137. ; 133:15
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Journal article (peer-reviewed)abstract
- The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently-labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding. The 2D Kd for L3-12 TCR binding to HLA-DQ8-glia-α1, 14±5 molecules/μm2, was only marginally influenced by including CD2 up to ∼200 bound molecules/μm2 but higher CD2 densities reduced the affinity up to 1.9-fold. Cell-SLB contact size increased steadily with ligand density without affecting binding for contacts up to ∼20% of total cell area but beyond this lamellipodia appeared, giving an apparent increase in bound receptors of up to 50%. Our findings show how parameters other than the specific protein-protein interaction can influence binding behavior at cell-cell contacts.
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| 6. |
- Junghans, Victoria, et al.
(author)
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Hydrodynamic trapping measures the interaction between membrane-associated molecules
- 2018
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1
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Journal article (peer-reviewed)abstract
- How membrane proteins distribute and behave on the surface of cells depends on the molecules’ chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method.
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| 7. |
- Jönsson, Peter, et al.
(author)
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Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions
- 2016
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 113:20, s. 5682-5687
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Journal article (peer-reviewed)abstract
- The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm2. This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.
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