31 research outputs found
A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity
The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host\u27s ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasit
Analysis of the CD200R family
Paired receptor families, consisting of multiple genetically and structurally similar but functionally opposite activating and inhibitory cell surface receptors, are among the fine tuners of the immune regulation. Recent studies on the evolutionary origin of these receptor families have suggested links to pathogen driven diversification, according to which activating receptors continuously evolve in order to counterbalance pathogens that try to subvert the immune response by stimulating the inhibitory receptor through their virulence factors.This thesis is about the CD200R paired receptor family. This family consists of an inhibitory receptor CD200R which is expressed on various leukocytes and delivers inhibitory signals upon engagement with its ligand CD200.In this study, the possibility that the activating members of the family evolved under pathogen pressure was investigated. Genomic DNA from twenty two different mice strains was screened for the presence of members of CD200R family. The number of activating receptors varied, CD200RLe and CD200RLc were found to be mutually exclusive and three strains possessed previously unknown members of CD200R family.In addition, the possibility that CD200R family members and other paired receptors interacted directly with bacteria was tested with a new assay but only the interaction of PIR-A1 with was found as previously reported.The rabbit CD200R family has been characterized and ligand receptor interaction between rabbit CD200 and rabbit CD200R has been demonstrated. However, no interaction between rabbit CD200R and a candidate viral CD200 homologue, the M141R protein of myxoma viruses, could be shown. This finding suggested a CD200R independent role for M141R molecule and possibly other homologues in pox viruses.Finally, two novel antibodies (OX131 and OX132) were characterized together with formerly generated antibodies against mouse CD200R family. The binding specificities and their effects on the CD200-CD200R interaction have been shown. This will help usage of these antibodies in various studies on the functionality and distribution of these receptors.</p
An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes
Analysis of the CD200R family
Paired receptor families, consisting of multiple genetically and structurally similar but functionally opposite activating and inhibitory cell surface receptors, are among the fine tuners of the immune regulation. Recent studies on the evolutionary origin of these receptor families have suggested links to pathogen driven diversification, according to which activating receptors continuously evolve in order to counterbalance pathogens that try to subvert the immune response by stimulating the inhibitory receptor through their virulence factors. This thesis is about the CD200R paired receptor family. This family consists of an inhibitory receptor CD200R which is expressed on various leukocytes and delivers inhibitory signals upon engagement with its ligand CD200. In this study, the possibility that the activating members of the family evolved under pathogen pressure was investigated. Genomic DNA from twenty two different mice strains was screened for the presence of members of CD200R family. The number of activating receptors varied, CD200RLe and CD200RLc were found to be mutually exclusive and three strains possessed previously unknown members of CD200R family. In addition, the possibility that CD200R family members and other paired receptors interacted directly with bacteria was tested with a new assay but only the interaction of PIR-A1 with <em)S. aureus was found as previously reported. The rabbit CD200R family has been characterized and ligand receptor interaction between rabbit CD200 and rabbit CD200R has been demonstrated. However, no interaction between rabbit CD200R and a candidate viral CD200 homologue, the M141R protein of myxoma viruses, could be shown. This finding suggested a CD200R independent role for M141R molecule and possibly other homologues in pox viruses. Finally, two novel antibodies (OX131 and OX132) were characterized together with formerly generated antibodies against mouse CD200R family. The binding specificities and their effects on the CD200-CD200R interaction have been shown. This will help usage of these antibodies in various studies on the functionality and distribution of these receptors.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
CD200RLc and CD200RLe can generate activating signals when triggered by specific mAb.
<p>(A) Flow cytometry plots showing expression of CD200RLc (left panel) and CD200RLe (right panel) (solid tinted lines) compared to the control mAb (dashed lines) on RBL.2H3 cells stably transduced with either CD200RLc or CD200RLe together with mouse DAP12. (B) RBL.2H3 cells, transduced with CD200RLc and DAP12, CD200RLe and DAP12 or mock vectors, were plated and soluble OX110, OX131, OX132 or no mAb were tested by overnight stimulation. The level of degranulation was measured by assaying β-hexosaminidase in the cell lysate and supernatants. Antibody stimulated groups were compared with no antibody control group with statistically significant results indicated by *** (p<0.001; representative of three experiments).</p
The CD200/CD200R interaction can be blocked by OX131 but not OX110 mAb.
<p>A) Biotinylated rCD4d3+4 (dashed line), CD200R(1) rCD4d3+d4 (solid black line) and CD200R(2) rCD4d3+4 (solid grey line) proteins were immobilized onto streptavidin coated CM5 chips (681, 726, 704 response units respectively). The changes in response units (RU) upon sequential injection of different soluble proteins (boxed and indicated by vertical dots) are shown. (Both antibodies were injected three consecutive times to ensure saturation on the immobilized proteins.) (B) Table showing the increase in response units upon injection of soluble CD200 compared to the pre-injection states for each flow cell. The values for the control rCD4d3+4 indicate the signal due to the high protein content of the CD200 sample.</p
Specificity of OX110, OX131 and OX132 mAb.
<p>Streptavidin coupled magnetic beads were coated with biotinylated chimeric proteins containing extracellular domains of members of the mouse CD200R family and rCD4d3+4 and a biotinylation site (as indicated on top of each column). The binding of the mAb indicated in each row was analyzed by flow cytometry. (A) Protein coating levels for each group of magnetic beads were tested by staining with OX68 (CD4 mAb) (tinted solid line) or OX21 (control mAb) (thin line). Flow cytometry plot named rCD4 d3+4 indicates coating level for biotinylated rCD4d3+4 only. (B–D) OX110 (B), OX131 (C) and OX132 (D) mAb were used to stain magnetic beads coated with the chimeric proteins indicated above each column (tinted solid line), or control beads coated with biotinylated rCD4d3+4 only (dashed line). Data are representative of three experiments.</p
CD200R and CD200RLc expression on mouse leukocytes.
<p>(A) <b>Left panel:</b> Gating strategy for macrophage and neutrophils in peritoneal aspirates of naive (top) and zymozan stimulated (bottom) mice. For each mouse CD11b+ cells were gated in CD11b-forward scatter plot (left) then in these populations Gr-1(−)F4/80(+) population was gated as resident macrophages (top right), Gr-1(+)F4/80(−) population was gated as neutrophils (bottom right), Gr1(+)F4/80(+) population was gated as inflammatory macrophages (bottom right). <b>Right panel:</b> expression of CD200R (top row) and CD200RLc (bottom row) in macrophage and neutrophil populations (tinted solid lines) compared to control mAb (dashed lines). (B) <b>Left Panel:</b> Gating strategy for different <i>in vitro</i> cultures of mouse bone marrow cells. Cells grown for mast and basophil differentiation were first gated for FcεRI expression in FcεRI- forward scatter plot (top left). FcεRI(+) cells were further gated as C-kit(+)CD49b(−) mast cells (top right) and C-kit(−) CD49b(+) basophils (top right) in C-kit-CD49b plot. Cells cultured in IL-5 supplemented media were gated as CD11b(+)Siglec F(+) eosinophils in CD11b-Siglec F plot (bottom left). Cells cultured in GMCSF supplemented media were gated as CD11c(+)MHC II(+) dendritic cells in CD11c-MHC II plot (top right). <b>Right panel:</b> Expression of CD200R (top row) and CD200RLc (bottom row) in <i>in vitro</i> cultures of mouse bone marrow cells (tinted solid lines) (C) <b>Left panel:</b> Gating strategy for lymphocytes derived from mouse spleen. B cells were gated for B220 expression in B220-forward scatter plot (top left). NK cells were gated as double positives in NK1.1-CD49b plot (top right). T cells were first gated as CD3(+) population in CD3-forward scatter plot (bottom left), then this population was further gated into CD4(+) and CD8(+) cells in CD4/CD8 plot (bottom right). <b>Right panel:</b> Expression of CD200R in unstimulated splenocytes (top row), expression of CD200RLc in unstimulated splenocytes (middle row) and expression of CD200R in <i>in vitro</i> stimulated splenocytes (LPS for B cells, CD3 mAb for T cells and IL-2 for NK cells) (bottom row) are shown (tinted solid lines) compared to control mAb (dashed lines).</p
Repertoire of CD200R family genes and predicted reactivity of mAb.
<p>(A) The different genes present in common mice strains from genomic and biochemical analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063325#pone.0063325-Wright1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063325#pone.0063325-Akkaya2" target="_blank">[16]</a>. (B) The predicted gene products recognised by the three mAb from this study.</p