scholarly journals Conformational variation between allelic variants of cell-surface ovine prion protein

2004 ◽  
Vol 381 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Alana M. THACKRAY ◽  
Sujeong YANG ◽  
Edmond WONG ◽  
Tim J. FITZMAURICE ◽  
Robert J. MORGAN-WARREN ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Lymphoid Tissues ◽  
Peripheral Blood Mononuclear ◽  
Variable Binding ◽  
Allelic Variants ◽  
Natural Scrapie ◽  
Resistant Genotypes

The distribution of prion infectivity and PrPSc between peripheral lymphoid tissues suggests their possible haematogenic spread during the progression of natural scrapie in susceptible sheep. Since ovine PBMCs (peripheral blood mononuclear cells) express PrPC, they have the potential to carry or harbour disease-associated forms of PrP. To detect the possible presence of disease-associated PrP on the surface of blood cells, an understanding is required of the conformations that normal ovine cell-surface PrPC may adopt. In the present study, we have used monoclonal antibodies that recognize epitopes in either the N- or C-terminal portions of PrP to probe the conformations of PrPC on ovine PBMCs by flow cytometry. Although PBMCs from scrapie-susceptible and -resistant genotypes of sheep expressed similar levels of cell-surface PrPC, as judged by their reactivity with N-terminal-specific anti-PrP monoclonal antibodies, there was considerable genotypic heterogeneity in the region between helix-1 and residue 171. Cells from PrP-VRQ (V136R154Q171) sheep showed uniform reactivity with monoclonal antibodies that bound to epitopes around helix-1, whereas cells from PrP-ARQ (A136R154Q171) and PrP-ARR (A136R154R171) sheep showed variable binding. The region between β-strand-2 and residue 171, which includes a YYR motif, was buried or obscured in cell-surface PrPC on PBMCs from scrapie-susceptible and -resistant sheep. However, an epitope of PrPC that is influenced by residue 171 was more exposed on PBMCs from PrP-VRQ sheep than on PBMCs from the PrP-ARQ genotype. Our results highlight conformational variation between scrapie-susceptible and -resistant forms of cell-surface PrPC and also between allelic variants of susceptible genotypes.

scholarly journals Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC

2006 ◽  
Vol 400 (2) ◽  
pp. 349-358 ◽  
Author(s):  
Alana M. Thackray ◽  
Tim J. Fitzmaurice ◽  
Lee Hopkins ◽  
Raymond Bujdoso
Keyword(s):  
Cell Surface ◽  
Mononuclear Cells ◽  
Genotypic Variation ◽  
Specific Monoclonal Antibody ◽  
Peripheral Blood Mononuclear ◽  
Protein Levels ◽  
Bovine Plasma ◽  
Natural Scrapie ◽  
Susceptibility And Resistance ◽  
Increased Susceptibility

Ovine PBMCs (peripheral blood mononuclear cells) express PrPC [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrPC expression by PBMCs, together with plasma PrPC levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrPC expression on normal ovine PBMCs by FACS with the presence of PrPC in plasma measured by capture–detector immunoassay. FACS showed similar levels of cell-surface PrPC on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrPC showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrPC levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrPC. Homozygous VRQ sheep showed the highest plasma PrPC level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrPC levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrPC expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrPC in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrPC was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrPC.

scholarly journals Modification of blood cell PrP epitope exposure during prion disease

2005 ◽  
Vol 390 (2) ◽  
pp. 563-571 ◽  
Author(s):  
Alana M. Thackray ◽  
Stephen J. Ryder ◽  
Raymond Bujdoso
Keyword(s):  
Cell Surface ◽  
Prion Disease ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Solvent Exposure ◽  
Peripheral Blood Mononuclear ◽  
Cell Surface Glycoprotein ◽  
Blood Mononuclear Cells

PrPC [normal cellular PrP (prion-related protein)] is a glycosylphosphatidylinositol-linked cell-surface glycoprotein that is expressed primarily by cells of the central and peripheral nervous system and the lymphoreticular system. During prion disease, PrPC undergoes structural modification to PrPSc (abnormal disease-specific conformation of PrP). The appearance of prion infectivity and PrPSc within different peripheral lymphoid tissue sites during natural scrapie infection in sheep is suggestive of haematogenic dissemination. For this to occur, blood cells may harbour or carry disease-associated PrP and in doing so present altered conformations of PrP on their cell-surface. In the present study, we show that changes in PrP epitope expression, or accessibility, can be detected on peripheral blood mononuclear cells during the course of experimental scrapie in susceptible sheep. Peripheral blood mononuclear cells isolated from VRQ homozygous lambs inoculated orally with scrapie were probed with either N- or C-terminal-specific anti-PrP monoclonal antibodies and analysed by flow cytometry. During the progression of scrapie, significant alterations were seen in the exposure of particular cell-surface PrP epitopes. These modifications included increased accessibility to N-terminal regions of the PrP molecule, to the region between β-strand-2 and residue 171, and to the C-terminal region of helix-3. Increased accessibility in the globular C-terminal domain of PrP occurred in the vicinity of tyrosine dimers, which are believed to have increased solvent exposure in disease-associated PrP. We suggest that the alterations in anti-PrP monoclonal antibody recognition of cell-surface PrP on blood cells from scrapie-infected sheep are indicative of structural changes within this molecule that may be relevant to prion disease.

scholarly journals HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Saintedym Wills ◽  
Kwan-Ki Hwang ◽  
Pinghuang Liu ◽  
S. Moses Dennison ◽  
Matthew Zirui Tay ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Functional Properties ◽  
Mononuclear Cells ◽  
Immunoglobulin A ◽  
Systemic Circulation ◽  
Peripheral Blood Mononuclear ◽  
Immune Mechanisms ◽  
Clonal Cell Lines ◽  
Hiv 1

ABSTRACTVaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments.IMPORTANCEHost-pathogen interactionsin vivoinvolve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the functional properties of HIV-1-specific IgG, more studies are needed on the functional attributes of HIV-1-specific IgA, specifically for vaccine-elicited IgA. Characterization of the functional properties of HIV-1 Env-specific IgA monoclonal antibodies from human vaccine clinical trials are critical toward understanding the capacity of the host immune response to block HIV-1 acquisition.

Abstract 20: Resolution Phenotype of Monocytes and Macrophages is Altered in Peripheral Arterial Disease

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Melinda S Schaller ◽  
Laura Menke ◽  
Mian Chen ◽  
Warren J Gasper ◽  
S. Marlene Grenon ◽  
...  
Keyword(s):  
Peripheral Arterial Disease ◽  
Cell Surface ◽  
Phagocytic Activity ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Arterial Disease ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Peripheral Blood Mononuclear ◽  
Peripheral Arterial

Introduction: Peripheral arterial disease (PAD) is a chronic disease characterized by systemic inflammation. Monocytes (Mo) and macrophages play a central role in vascular inflammation and its resolution. We hypothesize that impaired resolution in PAD results in poor clinical outcomes. Methods: Resolution phenotype was assessed by phagocytic activity of leukocytes, Mo cell surface markers, and cytokine profiling of Mo-derived macrophages (MDM). Phagocytosis and cell-surface markers were determined by flow cytometry. MDMs were generated from peripheral blood mononuclear cells via density gradient centrifugation. Cytokines were measured by ELISA following MDM differentiation and subsequent stimulation with LPS. Results: Circulating Mo and neutrophils (PMN) isolated from PAD patients (n=9) demonstrated significantly lower phagocytic activity (Mo: >30%, p<.001; PMN: >25%, p<.01, Fig. 1) as compared to healthy subjects (HS) (n=14). Cell-surface marker analysis demonstrated a higher proportion of the pro-inflammatory intermediate Mo subset (CD14 ++ 16 + , 1.8-fold, p=.04) in PAD compared to HS. MDM from PAD subjects retain their intrinsic inflammatory program by producing more IL-6 (PAD 3138±2676 ng/mL, HS 731±854 ng/mL p=.03) and IL-1β (PAD 244±236 ng/mL, HS 24.1±23.8 ng/mL p=.04) than those from HS. Upon stimulation with LPS, MDM from PAD subjects secrete more IL-6 (PAD 23353±22483 ng/mL, HS 5097±5836 ng/mL p=.05) than those from HS. Conclusions: Circulating Mo and PMN in patients with PAD have substantially lower phagocytic activity as well as a greater proportion of the pro-inflammatory intermediate Mo subset compared to HS. MDM preserve their elevated inflammatory state throughout culture and retain a heightened response upon latter stimulatory cues. Collectively these data demonstrate a heightened inflammatory and impaired resolution phenotype in PAD that has potential implications for disease progression and response to interventions.

Reduced Expression of Annexin A2 is Associated with Impaired Cell Surface Fibrinolysis and Venous Thromboembolism

Blood ◽  
2021 ◽  
Author(s):  
Hannah Fassel ◽  
Huigen Chen ◽  
Mary Ruisi ◽  
Neha Kumar ◽  
Maria T DeSancho ◽  
...  
Keyword(s):  
Risk Factor ◽  
Venous Thromboembolism ◽  
Cell Surface ◽  
Mononuclear Cells ◽  
Annexin A2 ◽  
Clot Lysis ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Intravascular Thrombosis

Reduced plasma fibrinolysis has been identified as a potential risk factor for venous thromboembolism (VTE), but the role of cell surface fibrinolysis in VTE is unknown. The annexin A2/S100A10 complex serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), augmenting plasmin generation by 60-fold on the endothelial cell surface. Several studies in both mice and humans support the concept that A2 regulates fibrin homeostasis and intravascular thrombosis in vivo. Here, we examined A2 protein expression and function in 115 adult subjects with venous thromboembolism (VTE) and 87 healthy controls. Using peripheral blood mononuclear cells (PBMCs) as a surrogate for endothelial cells, we found a 41% mean decrease in cell surface tPA-dependent fibrinolytic activity in subjects who had a positive personal and family history of VTE, but tested negative for known inherited thrombophilias. A2 protein was reduced on average by 70%, and mRNA levels by 30%, but neither decrease correlated with anticoagulant therapy. [Sentence omitted] Neither cell A2 protein nor cell surface plasmin generation correlated with plasma-based clot lysis times, suggesting that the plasma and cell surface fibrinolytic systems operate independently of one another. These data suggest that reduced expression of annexin A2 protein is associated with cell surface hypofibrinolysis and may represent a novel risk factor for inherited thrombophilia.

scholarly journals Sodium sulfite (SoS) as decontamination strategy for Fusarium-toxin contaminated maize and its impact on immunological traits in pigs challenged with lipopolysaccharide (LPS)

2020 ◽  
Vol 36 (4) ◽  
pp. 429-442
Author(s):  
Anh-Tuan Tran ◽  
Jeannette Kluess ◽  
Susanne Kersten ◽  
Andreas Berk ◽  
Marleen Paulick ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Sodium Sulfite ◽  
T Cell Subsets ◽  
Lymphoid Tissues ◽  
Fluorescence Signal ◽  
Polymorphonuclear Cells ◽  
Mesenteric Lymph Nodes ◽  
Peripheral Blood Mononuclear ◽  
The Impact

Abstract The main objective of this study was to evaluate the effects of sodium sulfite (SoS) treatment of maize and its impact on the porcine immune system in the presence of an LPS-induced systemic inflammation. Control maize (CON) and Fusarium-toxin contaminated maize (FUS) were wet-preserved (20% moisture) for 79 days with (+) or without (−) SoS and then included at 10% in a diet, resulting in four experimental groups: CON−, CON+, FUS−, and FUS+ with deoxynivalenol (DON) concentrations of 0.09, 0.05, 5.36, and 0.83 mg DON/kg feed, respectively. After 42-day feeding trial (weaned barrows, n = 20/group), ten pigs per group were challenged intraperitoneally with either 7.5 μg LPS/kg BW or placebo (0.9% NaCl), observed for 2 h, and then sacrificed. Blood, mesenteric lymph nodes, and spleen were collected for phenotyping of different T cell subsets, B cells, and monocytes. Phagocytic activity and intracellular formation of reactive oxygen species (ROS) were analyzed in both polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) using flow cytometry. Our results revealed that the impact of DON was more notable on CD3+CD4+CD8+ T cells in lymphoid tissues rather than in blood T cells. In contrast, SoS treatment of maize altered leukocyte subpopulations in blood, e.g., reduced the percentage and fluorescence signal of CD8high T cells. Interestingly, SoS treatment reduced the amount of free radicals in basal ROS-producing PMNs only in LPS-challenged animals, suggesting a decrease in basal cellular ROS production (pSoS*LPS = 0.022).

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