19 research outputs found
Investigating celiac disease using recombinant soluble MHC class II molecules
No full textCeliac disease (CD) is a chronic intestinal inflammation showing a strong association to HLA-DQ2.5. This thesis focused on the use of soluble MHC class II tetramers to examine the underlying mechanism for this selective CD association and also to develop a potential diagnostic tool based on the detection of gluten specific T cells.
DQ2.5 is associated with atypically high amounts of Ii-chain derived CLIP peptides. This phenotype was assigned to an abnormally low interaction with the peptide editing molecule HLA-DM, and high kinetic stability of CLIP peptides to DQ2.5. HLA-DQ2.2 is a highly similar molecule, but does not show strong CD association. Interestingly, this molecule displayed a CLIP low phenotype due to a drastically reduced kinetic stability for CLIP. This was assigned to a polymorphic residue causing the gain/loss of a hydrogen bond involved in peptide binding.
In addition, the application of a short bread challenge allowed for the detection of gluten reactive T cells in peripheral blood of CD patients using flow cytometry and MHC tetramers. This opens for the use of MHC tetramers as a diagnostic tool for diseases where the antigen and MHC restriction elements are known
The construction and analysis of low TCF11/Nrf1 expressing plasmids : regarding transactivational ability and intracellular localization
No full textTCF11 is a regulatory transcription factor belonging to the CNC-bZIP family. The specific biological function of this protein is still unknown. However, knockout studies in mice have revealed its importance during embryo development, and other studies have also displayed its involvement in the cell’s defense system against oxidants and carcinogens. The transactivating ability and intracellular localization of TCF11 and the isoform Nrf1 have been studied in cells using high expression plasmids. Due to the recent findings that over-expression of TCF11 in transfected cells caused an increase in cell mortality, the need for lower TCF11/Nrf1 expressing plasmids emerged. An additional reason for constructing the low expression plasmids was to study the localization and transactivating abilities of the proteins at levels closer to the endogenous situation. The transactivating ability was estimated by measuring the luciferase activity in COS-1 cells transiently co-transfected with a reporter plasmid and a high or low TCF11/Nrf1 expressing plasmid. The intracellular localization images were acquired by means of epifluorescence and confocal microscopy. The initial low expression constructs proved unsuitable due to the empty vector’s ability to cause indirect activation of the reporter plasmid. The second set of constructs were low expression plasmids that permitted verification of the nuclear detainment of Nrf1. However, further intracellular compartmentalization could not be detected for either Nrf1 or TCF11. In addition, TCF11 displayed a higher transactivating ability compared to Nrf1
Soluble T-cell receptors produced in human cells for targeted delivery.
No full textRecently, technology has become available to generate soluble T-cell receptors (sTCRs) that contain the antigen recognition part. In contrast to antibodies, sTCRs recognize intracellular in addition to extracellular epitopes, potentially increasing the number of applications as reagents for target detection and immunotherapy. Moreover, recent data show that they can be used for identification of their natural peptide ligands in disease. Here we describe a new and simplified expression method for sTCRs in human cells and show that these sTCRs can be used for antigen-specific labeling and elimination of human target cells. Four different TCRs were solubilized by expression of constructs encoding the TCR alpha (α) and beta (β) chains lacking the transmembrane and intracellular domains, linked by a ribosomal skipping 2A sequence that facilitates equimolar production of the chains. Cell supernatants containing sTCRs labeled target cells directly in a peptide (p)-human leukocyte antigen (HLA)-specific manner. We demonstrated that a MART-1p/HLA-A*02:01-specific sTCR fused to a fluorescent protein, or multimerized onto magnetic nanoparticles, could be internalized. Moreover, we showed that this sTCR and two sTCRs recognizing CD20p/HLA-A*02:01 could mediate selective elimination of target cells expressing the relevant pHLA complex when tetramerized to streptavidin-conjugated toxin, demonstrating the potential for specific delivery of cargo. This simple and efficient method can be utilized to generate a wide range of minimally modified sTCRs from the naturally occurring TCR repertoire for antigen-specific detection and targeting
The efficacy of emamectin benzoate against infestations of Lepeophtheirus salmonis on farmed Atlantic salmon (Salmo salar L) in Scotland, 2002-2006
No full textInfestations of the parasitic copepod Lepeophtheirus salmonis, commonly referred to as sea lice, represent a major challenge to commercial salmon aquaculture. Dependence on a limited number of theraputants to control such infestations has led to concerns of reduced sensitivity in some sea lice populations. This study investigates trends in the efficacy of the in-feed treatment emamectin benzoate in Scotland, the active ingredient most widely used across all salmon producing regions. Study data were drawn from over 50 commercial Atlantic salmon farms on the west coast of Scotland between 2002 and 2006. An epi-informatics approach was adopted whereby available farm records, descriptive epidemiological summaries and statistical linear modelling methods were used to identify factors that significantly affect sea lice abundance following treatment with emamectin benzoate (SLICEH, Schering Plough Animal Health). The results show that although sea lice infestations are reduced following the application of emamectin benzoate, not all treatments are effective. Specifically there is evidence of variation across geographical regions and a reduction in efficacy over time. Reduced sensitivity and potential resistance to currently available medicines are constant threats to maintaining control of sea lice populations on Atlantic salmon farms. There is a need for on-going monitoring of emamectin benzoate treatment efficacy together with reasons for any apparent reduction in performance. In addition, strategic rotation of medicines should be encouraged and empirical evidence for the benefit of such strategies more fully evaluated
Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation
No full text10.1038/ni.1780Nature Immunology10101096-1101NIAM
DMF5 sTCR is internalized upon specific ligand binding.
No full text<p>(a) HeLa cells transfected with either SCT-M1-mCherry or SCT-CD20-mCherry (red) were incubated with His-tagged DMF5 sTCR labeled with an anti-His antibody and visualized using anti-mouse AF488 (green). Co-localization of the sTCR and SCT-M1 is shown in yellow (arrow). (b) Sup-T1 cells expressing SCT-M1 were incubated with DMF5 supernatants containing monomeric sTCR mCherry-His at 37°C for 30 minutes. The cells were subsequently put on ice to block endocytosis and stained with anti-His-AF647 (His). The nucleus was visualized by Hoechst stain (blue). (c) Sup-T1 cells expressing SCT-M1 were incubated with biotinylated DMF5 sTCR-mCherry bound to SA-Miltenyi nanobeads at 37°C.</p
Multimerization of the sTCR increases sensitivity of antigen detection by flow cytometry.
No full text<p>(a) SupT1 cells expressing SCT-CD20 were incubated with increasing concentrations of CD20 swap-sTCR monomer or tetramer, as indicated, and indirectly labeled with anti-FLAG AF647. Results shown are representative of two experiments, and error bars represent SD of duplicates (b) HLA-A2<sup>+</sup> SupT1 cells were loaded with indicated concentrations of peptide and subsequently labeled with saturating amounts of either sTCR tetramer or nanobeads conjugated to sTCR monomers, followed by staining with anti-FLAG-AF647. Results shown are representative of two experiments. SupT1 cells expressing SCT-CD20 were used as a positive control. The scale shows the calculation of Arcsinh ratio of the Median. (c) SupT1 cells were loaded with increasing concentrations of CD20p (left panel), MART-1p or WT MART-1p (right panel), followed by labeling with saturating amounts of indicated sTCR tetramer, and indirectly stained with anti-FLAG AF647. Irrelevant peptide control (irr peptide) was used at 100 μM (disconnected symbols). Results shown are from one experiment representative of 3 (CD20p and MART-1p) or 2 (WT MART-1p) experiments. Error bars indicate SD of duplicates. (d) SupT1 cells expressing SCT-M1 were incubated with increasing concentrations of PE-conjugated sTCR tetramer.</p
Complexes of Two Cohorts of CLIP Peptides and HLA-DQ2 of the Autoimmune DR3-DQ2 Haplotype Are Poor Substrates for HLA-DM
No full textAntigen-specific elimination of target cells by sTCR-Saporin as measured by <sup>3</sup>H-thymidine incorporation.
No full text<p>(a) SupT1 cells constitutively expressing SCT-M1 or SCT-CD20 were incubated with or without (NT) 10–40 nM DMF5 sTCR-Sap for 3 days. Cells were then labeled with <sup>3</sup>H-thymidine and radioactive incorporation was measured (cpm). (b) SupT1 cells expressing SCT-M1 were incubated with increasing amounts of DMF5 sTCR-Sap for 3 days and <sup>3</sup>H-thymidine incorporation was plotted as in (a). (c) HEK293 cells were pulsed with or without 10 μM MART-1p in the presence or absence of 20 nM DMF5 sTCR-Sap for 3 days, and <sup>3</sup>H-thymidine incorporation was determined. The experiments shown in (a-c) are each representative of two performed and error bars represent SD from triplicates.</p
