Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial ...PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2-4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF
EVs.
Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles.
To investigate ...reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles.
Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, alpha-enolase peptide-1 and the C1-epitope of type II collagen (C1(III)). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed.
72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients' sera and in 41% for both citrullinated alpha-enolase peptide-1 and citrullinated C1(III). These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles.
Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.
Organisms have to be sufficiently robust to environmental and genetic perturbations, yet plastic enough to cope with stressful scenarios to which they are not fully adapted. How this apparent ...conflict between robustness and plasticity is resolved at the cellular and whole organism levels is not clear. Here we review and discuss evidence in flies suggesting that the environment can modulate the balance between robustness and plasticity. The outcomes of this modulation can vary from mild sensitizations that are hardly noticeable, to overt qualitative changes in phenotype. The effects could be at both the cellular and whole organism levels and can include cellular de-/trans-differentiation (‘Cellular reprogramming’) and gross disfigurements such as homeotic transformations (‘Tissue/whole organism reprogramming’). When the stress is mild enough, plastic changes in some processes may prevent drastic changes in more robust traits such as cell identity and tissue integrity. However, when the stress is sufficiently severe, this buffering may no longer be able to prevent such overt changes, and the resulting phenotypic variability could be subjected to selection and might assist survival at the population level. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.
•Relations between stress, developmental plasticity, and robustness are insufficiently understood.•Here we review and discuss relevant examples and mechanisms in flies.•We suggest that stress can mediate the balance between robustness and plasticity.•We argue that plastic responses may act to prevent changes in more robust traits.
Proinflammatory CD4+CD28null T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to ...identify functional differences between CD4+CD28null T cells from blood and synovial fluid in comparison with conventional CD28‐expressing CD4+ T cells. Forty‐four patients with RA, displaying a distinct CD4+CD28null T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN‐γ, TNF, IL‐17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4+CD28null T cells were significantly more hypomethylated in the CNS‐1 region of the IFNG locus than conventional CD4+CD28+ T cells and produced higher levels of both IFN‐γ and TNF after TCR cross‐linking. CD4+CD28null T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL‐17A production could hardly be detected in CD4+CD28null cells from the blood, a significant production was observed in CD4+CD28null T cells from synovial fluid. CD4+CD28null T cells were not only found to differ from conventional CD4+CD28+ T cells in the circulation, but we could also demonstrate that synovial CD4+CD28null T cells showed additional effector functions (IL‐17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28null population.
A hallmark of celiac disease is autoantibodies to transglutaminase 2 (TG2). By visualizing TG2-specific antibodies by antigen staining of affected gut tissue, we identified TG2-specific plasma cells ...in the lamina propria as well as antibodies in the subepithelial layer, inside the epithelium, and at the brush border. The frequency of TG2-specific plasma cells were found not to correlate with serum antibody titers, suggesting that antibody production at other sites may contribute to serum antibody levels. Upon commencement of a gluten-free diet, the frequency of TG2-specific plasma cells in the lesion dropped dramatically within 6 months, yet some cells remained. The frequency of TG2-specific plasma cells in the celiac lesion is thus dynamically regulated in response to gluten exposure. Laser microdissection of plasma cell patches, followed by antibody gene sequencing, demonstrated that clonal cells were seeded in distinct areas of the mucosa. This was confirmed by immunoglobulin heavy chain repertoire analysis of plasma cells isolated from individual biopsies of two untreated patients, both for TG2-specific and non-TG2-specific cells. Our results shed new light on the processes underlying the B-cell response in celiac disease, and the approach of staining for antigen-specific antibodies should be applicable to other antibody-mediated diseases.
Many genetic variants associate with the risk of developing rheumatoid arthritis (RA); however, their functional roles are largely unknown. Here, we aimed to investigate whether the RA-associated ...serotonin receptor 2A (HTR2A) haplotype affects T-cell and monocyte functions. Patients with established RA (n=379) were genotyped for two single-nucleotide polymorphisms (SNPs) in the HTR2A locus, rs6314 and rs1328674, to define presence of the risk haplotype for each individual. Patients with and without the RA-associated TC haplotype were selected and T-cell and monocyte function was monitored following in vitro stimulations with staphylococcal enterotoxin B and lipopolysaccharide (LPS) using multiparameter flow cytometry. Within the cohort, 44 patients were heterozygous for the TC haplotype (11.6%) while none were homozygous. Upon stimulation, T cells from TC-carrier patients produced more proinflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-17 (IL-17) and interferon gamma (IFN-γ)) and monocytes produced higher levels of TNF-α compared with patients carrying the non-TC haplotype (P<0.05 and 0.01, respectively). Such cytokine production could be inhibited in the presence of the selective 5-HT2 receptor agonist (2,5-Dimethoxy-4-iodoamphetamine, DOI); interestingly, this effect was more pronounced in TC carriers. Our data demonstrate that association of RA with a distinct serotonin receptor haplotype has functional impact by affecting the immunological phenotype of T cells and monocytes.
Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we ...studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19
PC subset was dynamically exchanged, whereas of two CD19
PC subsets, CD45
PCs exhibited little and CD45
PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45
PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19
PC subsets support selection and maintenance of protective PCs for life in human intestine.
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