Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

The authors thank the UK EPSRC the CR-UK/EPSRC/MRC/DoH (England) imaging Program and the European Union project FAMOS (FP7 ICT, contract no. 317744) for funding, K.D. is a Royal Society-Wolfson Merit Award Holder.Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.Publisher PD

The genetic modification of a rat class I major histocompatibility complex antigen

SIGLEAvailable from British Library Document Supply Centre- DSC:D61143 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

The Oxidative Folding and Misfolding of Human Leukocyte Antigen-B27

The major histocompatibility complex class I molecule human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies. Many autoimmune diseases exhibit associations with major histocompatibility complex molecules encoded within the class II locus with defined immune responses either mediated by T or B-lymphocytes. Despite the association being known for over 30 years, no defined immune response and target autoantigens have been characterized for the spondyloarthropathies. Thus, the mechanism and role of HLA-B27 in disease pathogenesis remains undetermined. One hypothesis that has recently received much attention has focused around the enhanced propensity for HLA-B27 to misfold and the increased tendency of the heavy chain to dimerize. The misfolding of HLA-B27 has been associated with its redox status and this is postulated to be involved in disease development. Here we discuss the impact of the redox status on HLA-B27 biosynthesis and functio

Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical

The MHC Class I Heavy Chain Structurally Conserved Cysteines 101 and 164 Participate in HLA-B27 Dimer Formation

The human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies (SpAs). The unusual biochemistry of HLA-B27 has been proposed to participate in disease development, especially the enhanced ability of HLA-B27 to form several heavy chain-dimer populations. HLA-B27 possesses three unpaired cysteine (C) residues at position 67, 308, and 325, in addition to the four conserved cysteine residues at p101, 164, 203, and 259. C67 was proposed to participate in dimer formation of recombinant HLA-B27 protein and in vivo heavy chain-dimers. However, the structurally conserved C164 was demonstrated to participate in endoplasmic reticulum (ER) resident heavy chain-dimer formation. We therefore wanted to determine whether these aggregates involve cysteines other than C164 and the basis for the difference between the observed heavy chain-dimer species. Results: We determined that C164 and C101 can form distinct dimer structures and that the heterogenous nature of heavy chain-dimer species is due to differences in both redox status and conformation. Different HLA-B27 dimer populations can be found in physiologically relevant cell types derived from HLA-B27-positive patients with inflammatory arthritis. In addition, HLA-B27 dimer formation can be correlated with cellular stress induction. Innovation: The use of both mutagenesis and manipulating cellular redox environments demonstrates that HLA-B27 dimerization requires both specific cysteine–cysteine interactions and conformations with differing redox states. Conclusion: HLA-B27 heavy chain-dimerization is a complex process and these findings provide an insight into HLA-B27 misfolding and a potential contribution to inflammatory disease development