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

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.

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

Biochemical Journal ◽

10.1042/bj20040351 ◽

2004 ◽

Vol 381 (1) ◽

pp. 221-229 ◽

Cited By ~ 15

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.

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The CD40 rs1883832 Polymorphism Affects Sepsis Susceptibility and sCD40L Levels

BioMed Research International ◽

10.1155/2018/7497314 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Zuo-Liang Liu ◽

Jing Hu ◽

Xue-Fei Xiao ◽

Yue Peng ◽

Shang-Ping Zhao ◽

...

Keyword(s):

Mononuclear Cells ◽

Healthy Controls ◽

Peripheral Blood Mononuclear ◽

Single Nucleotide ◽

Protein Levels ◽

Potential Association ◽

Functional Single Nucleotide Polymorphism ◽

The Relationship ◽

Control Study ◽

Increased Susceptibility

Sepsis is a severe and progressive disease characterized by systemic inflammatory response syndrome (SIRS). CD40 serves as a vital link between immune response and inflammation. This study was designed to investigate the potential association between a functional single-nucleotide polymorphism (SNP) of CD40 (rs1883832) and susceptibility to sepsis. We first performed a case-control study to explore the relationship between the CD40 rs1883832 polymorphism and sepsis. CD40 mRNA expression and protein expression were determined by real-time PCR and western blotting, respectively, in peripheral blood mononuclear cells (PBMCs) from sepsis patients and healthy controls. The plasma sCD40L levels in the two groups were measured by ELISA. The results showed that the frequencies of the TT genotype and the CD40 rs1883832 T allele were significantly higher in sepsis patients than in healthy controls. Plasma sCD40L levels were also significantly increased in sepsis patients. In addition, TT genotype carriers among sepsis patients displayed the highest CD40 expression at both the mRNA and protein levels, accompanied by the highest plasma sCD40L concentrations. In conclusion, the CD40 rs1883832 T allele acts as a risk factor for increased susceptibility to sepsis and may be involved in the process of sepsis through regulation of CD40 expression and plasma sCD40L levels.

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Peripheral Blood Mononuclear Cells Antioxidant Adaptations to Regular Physical Activity in Elderly People

Nutrients ◽

10.3390/nu10101555 ◽

2018 ◽

Vol 10 (10) ◽

pp. 1555 ◽

Cited By ~ 5

Author(s):

Carla Busquets-Cortés ◽

Xavier Capó ◽

Maria Bibiloni ◽

Miquel Martorell ◽

Miguel Ferrer ◽

...

Keyword(s):

Physical Activity ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Regular Physical Activity ◽

Antioxidant Defenses ◽

Active Lifestyle ◽

Peripheral Blood Mononuclear ◽

Protein Levels ◽

Blood Mononuclear Cells

Regular physical activity prescription is a key point for healthy aging and chronic disease management and prevention. Our aim was to evaluate the antioxidant defense system and the mitochondrial status in peripheral blood mononuclear cells (PBMCs) and the level of oxidative damage in plasma in active, intermediate and inactive elderly. In total, 127 healthy men and women >55 years old participated in the study and were classified according on their level of declared physical activity. A more active lifestyle was accompanied by lower weight, fat mass and body mass index when compared to a more sedentary life-style. Active participants exhibited lower circulating PBMCs than inactive peers. Participants who reported higher levels of exercise had increased antioxidant protein levels when compared to more sedentary partakers. Carbonylated protein levels exhibited similar behavior, accompanied by a significant raise in expression of cytochrome c oxidase subunit IV in PBMCs. No significant changes were found in the activities of antioxidant enzymes and in the expression of structural (MitND5) and mitochondrial dynamic-related (PGC1α and Mitofusins1/2.) proteins. Active lifestyle and daily activities exert beneficial effects on body composition and it enhances the antioxidant defenses and oxidative metabolism capabilities in PBMCs from healthy elderly.

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H3K23/H3K36 hypoacetylation and HDAC1 up-regulation are associated with adverse consequences in obstructive sleep apnea patients

Scientific Reports ◽

10.1038/s41598-021-00052-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yung-Che Chen ◽

Po-Yuan Hsu ◽

Chien-Hung Chin ◽

Chang-Chun Hsiao ◽

Chia-Wei Liou ◽

...

Keyword(s):

Obstructive Sleep Apnea ◽

Sleep Apnea ◽

Mononuclear Cells ◽

Gene Promoter ◽

Peripheral Blood Mononuclear ◽

Protein Levels ◽

Reactive Protein ◽

Primary Snoring ◽

Obstructive Sleep

AbstractThe aim of this study is to determine the roles of global histone acetylation (Ac)/methylation (me), their modifying enzymes, and gene-specific histone enrichment in obstructive sleep apnea (OSA). Global histone modifications, and their modifying enzyme expressions were assessed in peripheral blood mononuclear cells from 56 patients with OSA and 16 matched subjects with primary snoring (PS). HIF-1α gene promoter-specific H3K36Ac enrichment was assessed in another cohort (28 OSA, 8 PS). Both global histone H3K23Ac and H3K36Ac expressions were decreased in OSA patients versus PS subjects. H3K23Ac expressions were further decreased in OSA patients with prevalent hypertension. HDAC1 expressions were higher in OSA patients, especially in those with excessive daytime sleepiness, and reduced after more than 6 months of continuous positive airway pressure treatment. H3K79me3 expression was increased in those with high C-reactive protein levels. Decreased KDM6B protein expressions were noted in those with a high hypoxic load, and associated with a higher risk for incident cardiovascular events or hypertension. HIF-1α gene promoter-specific H3K36Ac enrichment was decreased in OSA patients versus PS subjects. In vitro intermittent hypoxia with re-oxygenation stimuli resulted in HDAC1 over-expression and HIF-1α gene promoter-specific H3K36Ac under-expression, while HDAC1 inhibitor, SAHA, reversed oxidative stress through inhibiting NOX1. In conclusions, H3K23/H3K36 hypoacetylation is associated with the development of hypertension and disease severity in sleep-disordered breathing patients, probably through up-regulation of HDAC1, while H3K79 hypermethylation is associated with higher risk of cardiovascular diseases, probably through down-regulation of KDM6B.

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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.20 ◽

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.

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

Blood ◽

10.1182/blood.2020008123 ◽

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.

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Metabolic Syndrome Is Associated with Oxidative Stress and Proinflammatory State

Antioxidants ◽

10.3390/antiox9030236 ◽

2020 ◽

Vol 9 (3) ◽

pp. 236 ◽

Cited By ~ 9

Author(s):

Margalida Monserrat-Mesquida ◽

Magdalena Quetglas-Llabrés ◽

Xavier Capó ◽

Cristina Bouzas ◽

David Mateos ◽

...

Keyword(s):

Oxidative Stress ◽

Metabolic Syndrome ◽

Vascular Function ◽

Mononuclear Cells ◽

Antioxidant Defenses ◽

Peripheral Blood Mononuclear ◽

Protein Levels ◽

Inflammatory Parameters ◽

Increased Risk ◽

Factor Α

Metabolic syndrome (MetS) is associated with increased risk of developing diabetes and cardiovascular diseases. MetS is also characterized by an increase of oxidative stress which contributes to impaired inflammation, vascular function, and atherosclerosis. The aim was to assess the oxidative stress and inflammatory markers in plasma and PBMCs in adults with or without MetS. Antioxidant and inflammatory parameters were measured in peripheral blood mononuclear cells (PBMCs) of 80 men and 80 women over 55 to 80-years-old residing in the Balearic Islands without previously documented cardiovascular disease. Circulating leukocytes, neutrophils, lymphocytes, basophils, and monocytes were higher in MetS subjects with respect to those without MetS. Plasma levels of malondialdehyde, tumor necrosis factor α (TNFα), and interleukin 6 (IL-6) levels were higher in MetS subjects in both genders, but the superoxide dismutase activity was lower. The myeloperoxidase plasma activity was higher in the MetS male subjects. Higher activities and protein levels of catalase and glutathione reductase in PBMCs were observed in MetS subjects in both genders. Obtained data show that MetS is associated with oxidative stress and a proinflammatory state and with high antioxidant defenses in PBMCs probably derived from a pre-activation state of immune cells.

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Cyclin E overexpression in relapsed adult acute lymphoblastic leukemias of B-cell lineage

Blood ◽

10.1182/blood.v87.8.3360.bloodjournal8783360 ◽

1996 ◽

Vol 87 (8) ◽

pp. 3360-3367 ◽

Cited By ~ 39

Author(s):

R Scuderi ◽

KA Palucka ◽

K Pokrovskaja ◽

M Bjorkholm ◽

KG Wiman ◽

...

Keyword(s):

Cell Cycle ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Lymphoblastic Leukemia ◽

Cyclin E ◽

Cell Lineage ◽

Cyclin A ◽

Mean Value ◽

Peripheral Blood Mononuclear ◽

Protein Levels

Using Western blot analysis, we examined cyclin E and cyclin A protein levels in 19 patients with acute lymphoblastic leukemia ([ALL] 15 B-ALL and four T-ALL). Whereas normal, nonproliferating peripheral blood mononuclear cells (PBMCs) expressed low levels of the 50-kD cyclin E, ALL blasts in the peripheral blood, although showing low-level or no proliferation as judged by FACS/cell-cycle analysis and cyclin A protein levels, expressed high levels of cyclin E, with a mean value similar to that of the proliferating Burkitt's lymphoma cell line, Akata. The accumulation of a protein shown to shorten the G1 phase of the cell cycle, cyclin E, in growth-delayed leukemic blasts may reflect the malignant status of these cells. Before treatment, B-ALL cells expressed predominantly the 50-kD cyclin E. T-ALL samples displayed the 50-kD cyclin E protein and a smaller, approximately 43-kD cyclin E species. In paired B-ALL samples taken before treatment and at relapse, we found a significant overexpression of the 50-kD protein in relapsed samples (P < .006), plus the presence of up to four additional smaller- molecular-weight species of cyclin E, illustrating clear diagnosis versus relapse differences. Cyclin E expression in ALL blasts may correlate to the relative malignant status of the cells, with higher protein levels reflecting a more advanced stage of the disease and a greater potential to proliferate under permissive conditions.

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Frataxin deficiency in Friedreich’s ataxia is associated with reduced levels of HAX-1, a regulator of cardiomyocyte death and survival

Human Molecular Genetics ◽

10.1093/hmg/ddz306 ◽

2020 ◽

Vol 29 (3) ◽

pp. 471-482

Author(s):

Francesca Tiano ◽

Francesca Amati ◽

Fabio Cherubini ◽

Elena Morini ◽

Chiara Vancheri ◽

...

Keyword(s):

Pancreatic Beta Cells ◽

Mononuclear Cells ◽

Premature Death ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Peripheral Blood Mononuclear ◽

Protein Levels ◽

Potential Biomarker ◽

Mrna And Protein Expression ◽

Defence Proteins

Abstract Frataxin deficiency, responsible for Friedreich’s ataxia (FRDA), is crucial for cell survival since it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. In FRDA, the heart is frequently affected with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and cause premature death. A microarray analysis performed on FRDA patient’s lymphoblastoid cells stably reconstituted with frataxin, indicated HS-1-associated protein X-1 (HAX-1) as the most significantly upregulated transcript (FC = +2, P < 0.0006). quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and western blot analysis performed on (I) HEK293 stably transfected with empty vector compared to wild-type frataxin and (II) lymphoblasts from FRDA patients show that low frataxin mRNA and protein expression correspond to reduced levels of HAX-1. Frataxin overexpression and silencing were also performed in the AC16 human cardiomyocyte cell line. HAX-1 protein levels are indeed regulated through frataxin modulation. Moreover, correlation between frataxin and HAX-1 was further evaluated in peripheral blood mononuclear cells (PBMCs) from FRDA patients and from non-related healthy controls. A regression model for frataxin which included HAX-1, group membership and group* HAX-1 interaction revealed that frataxin and HAX-1 are associated both at mRNA and protein levels. Additionally, a linked expression of FXN, HAX-1 and antioxidant defence proteins MnSOD and Nrf2 was observed both in PBMCs and AC16 cardiomyocytes. Our results suggest that HAX-1 could be considered as a potential biomarker of cardiac disease in FRDA and the evaluation of its expression might provide insights into its pathogenesis as well as improving risk stratification strategies.

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