Increased Lipid Peroxidation May Be Linked to Ferritin Levels Elevation in Adult-Onset Still’s Disease

Lipid peroxidation (LPO) and hyper-ferritinemia are involved in inflammatory responses. Although hyper-ferritinemia is a characteristic of AOSD, its link to LPO remains unclear. We investigated the association between LPO and ferritin expression, and evaluated the relationship between LPO-related metabolites and inflammatory parameters. Mean fluorescence intensity (MFI) of LPO (C11-Biodipy581/591)-expressing PBMCs/monocytes in AOSD patients and healthy control (HC) subjects was determined by flow-cytometry analysis. Expression of ferritin and cytokines on PBMCs/macrophages was examined by immunoblotting. Plasma levels of LPO-related metabolites and cytokines were determined by ELISA and the MULTIPLEX platform, respectively. LPO MFI on PBMCs/monocytes were significantly higher in patients (median 4456 and 9091, respectively) compared with HC (1900, p < 0.05, and 4551, p < 0.01, respectively). Patients had higher ferritin expression on PBMCs (mean fold, 1.02) than HC (0.55, p < 0.05). Their ferritin expression levels on PBMCs stimulated with LPO inducers erastin or RSL3 (2.47 or 1.61, respectively) were higher than HC (0.84, p < 0.05, or 0.74, p < 0.01). Ferritin expression on erastin-treated/IL-1β-treated macrophages from patients were higher than those from HC (p < 0.001). The elevated levels of LPO-related metabolites, including malondialdehyde and 4-hydroxyalkenals, were positively correlated with disease activity scores, suggesting LPO involvement in AOSD pathogenesis. Increased ferritin expression on PBMCs/macrophages stimulated with LPO inducers indicates a link between LPO and elevated ferritin.

Sources of Variability in the Response of Labeled Microspheres and B Cells during the Analysis by a Flow Cytometer

A stochastic model of the flow cytometer measurement process was developed to assess the nature of the observed coefficient of variation (CV%) of the mean fluorescence intensity (MFI) from a population of labeled microspheres (beads). Several sources of variability were considered: the total number of labels on a bead, the path through the laser beam, the optical absorption cross-section, the quantum yield, the numerical aperture of the collection optics, and the photoelectron conversion efficiency of the photomultiplier (PMT) cathode. The variation in the number of labels on a bead had the largest effect on the CV% of the MFI of the bead population. The variation in the path of the bead through the laser beam was minimized using flat-top lasers. The variability in the average optical properties of the labels was of minor importance for beads with sufficiently large number of labels. The application of the bead results to the measured CV% of labeled B cells indicated that the measured CV% was a reliable measure of the variability of antibodies bound per cell. With some modifications, the model can be extended to multicolor flow cytometers and to the study of CV% from cells with low fluorescence signal.

Positive influence of aminosilanes on anti-EpCAM antibody immobilization on a glass surface

Immobilization of antibodies has a number of promising applications, including detection of biomolecules and cells. Well-oriented antibodies are required to bind them effectively. To eliminate the problem of random antibodies’ orientation, the surface of the device can be modified with silanes. This study aimed at elucidating if selected aminosilanes were able to bind antibodies in the appropriate orientation and thus retain their binding activity. Silanization of glass slides was performed using three amino-functional trialkoxysilanes – A, AE, and AEE. The immunofluorescent reaction was used to evaluate the potential of the silanized glass surface to bind anti-EpCAM antibodies. The affinity of selected anti-EpCAM HEA125 antibodies labeled with fluorochrome to tested silanized surfaces was evaluated by measuring the mean fluorescence intensity (MFI) in each analyzed area. The presented silanes effectively bound antibodies. Higher fluorescence intensity was noticed in the case of silane-coated glass slides in comparison to unmodified ones. The differences in the contact angles also confirmed this result. In the case of silane A, the fluorescence intensity reflected the amount of bound antibodies. However, there was no such a relation in the case of the silanes AE and AEE. Although our research gave promising results, the usefulness of selected silanes needs to be confirmed by further studies using cancer cells. Running title: Aminosilanes as enhancers of antibody immobilization

CD33 as a Leukocyte-Associated Marker Expressed on Human Spermatozoa

Background Sialic acid-binding immunoglobulin-type lectins (Siglecs) are commonly present on immune cells and often mediate cell-to-cell interactions and signaling. Studies have shown the presence of Siglecs 1, 2, 5, 6, 10 and 14 on human spermatozoa. To the best of our knowledge, the expression of CD33 on spermatozoa has not yet been studied. Methods Semen samples were collected from 25 healthy men with normal semen status. CD33 expression on purified spermatozoa was evaluated by flow cytometry methods. Results The results demonstrate the expression of CD33 on the surface of purified spermatozoa. The mean (± SD) of MFI (mean fluorescence intensity) was 12.85 (± 1.33) and the mean percentage of spermatozoa that express CD33 was 73.75 (± 3.75). Conclusion Results were obtained showing that spermatozoa express CD33 (or Siglec-3) on their surface. The physiological role of these molecules on spermatozoa remains to be determined. It is recommended that further research should be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis.

Apheresis Efficacy and Tolerance in the Setting of HLA-Incompatible Kidney Transplantation

Nearly 18% of patients on a waiting list for kidney transplantation (KT) are highly sensitized, which make access to KT more difficult. We assessed the efficacy and tolerance of different techniques (plasma exchanges [PE], double-filtration plasmapheresis [DFPP], and immunoadsorption [IA]) to remove donor specific antibodies (DSA) in the setting of HLA-incompatible (HLAi) KT. All patients that underwent apheresis for HLAi KT within a single center were included. Intra-session and inter-session Mean Fluorescence Intensity (MFI) decrease in DSA, clinical and biological tolerances were assessed. A total of 881 sessions were performed for 45 patients: 107 DFPP, 54 PE, 720 IA. The procedures led to HLAi KT in 39 patients (87%) after 29 (15–51) days. A higher volume of treated plasma was associated with a greater decrease of inter-session class I and II DSA (p = 0.04, p = 0.02). IA, PE, and a lower maximal DSA MFI were associated with a greater decrease in intra-session class II DSA (p < 0.01). Safety was good: severe adverse events occurred in 17 sessions (1.9%), more frequently with DFPP (6.5%) p < 0.01. Hypotension occurred in 154 sessions (17.5%), more frequently with DFPP (p < 0.01). Apheresis is well tolerated (IA and PE > DFPP) and effective at removing HLA antibodies and allows HLAi KT for sensitized patients.

Phenotypic Changes in T Cell and Macrophage Subtypes in Perivascular Adipose Tissues Precede High-Fat Diet-Induced Hypertension

Graphical AbstractSex-differences in immune cell activation status (numbers/mean fluorescence intensity) in MRPVAT (A) and APVAT (B). (Key: e.g., at 10 weeks, higher density/MFI of M1-like macrophages occur in CD females vs. CD males in MRPVAT.) Differences in mean arterial pressure between HFD and respective CD-fed rats are presented as mm Hg [Supplementary Figure 1 and as measured by radiotelemetry (Fernandes et al., 2018)] with 10, 17, and 24 weeks on diet.

Increased Expression of PAD4 in Neutrophils of MPO-ANCA Associated Vasculitis and its Correlation with the Vasculitis Activity

Myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (MPO-AAV) is the most common ANCA associated vasculitis (AAV) in china and other East Asian countries. Its pathogenesis remained elusive. We evaluated the expression of peptidylarginine deiminase (PAD4) in neutrophils from MPO-AAV and explored the relationship between the expression and the vasculitis activity, and also investigated the pathogenic mechanism. 40 patients initially diagnosed with MPO-AAV in the active state and 40 comparable healthy controls (HC) were enrolled in this study. The patient disease activity were evaluated with Birmingham Vasculitis Activity Index version 3 (BVAS-V3). The expression of PAD4 in their neutrophils, which was described as percentage of PAD4+ neutrophils in all neutrophils and mean fluorescence intensity (MFI) of PAD4+ neutrophils, was measured by flow cytometry (FCM). Their serum level of NETs, C5a and MPO-ANCA were detected using enzyme linked immunosorbent assay (ELISA). The percentage of PAD4+ neutrophils, MFI of PAD4+ neutrophils, the levels of serum NETs and C5a were markedly higher in patient group than that in HC group. Meanwhile, in patient group, the percentage of PAD4+ neutrophils, MFI of PAD4+ neutrophils and MPO-ANCA presented significantly correlation with NETs. Although univariable analysis showed the percentage of PAD4+ neutrophils, MFI of PAD4+ neutrophils, NETs, C5a and MPO-ANCA had respective correlation with BVAS, multivariable analysis indicated that only the percentage of PAD4+ neutrophils as well as the level of serious MPO-ANCA owned independent correlations with BVAS.PAD4 might be a new potential target for MPO-AAV treatment in the future.

Galectin-9 expression correlates with therapeutic effect in rheumatoid arthritis

Galectin-9 (Gal-9) is a multifunctional immunomodulatory factor highly expressed in RA. This study aimed to investigate the expression of Gal-9 and its correlation with disease activity and therapeutic response in RA patients. Active RA patients were enrolled and treated with tacrolimus (TAC) alone or in combination therapy for 12 weeks in a prospective cohort study. Clinical and immunological parameters were recorded at baseline and week 12. We measured Gal-9 expression in different T cell subsets and in plasma. The disease activity of RA patients decreased after treatment. At baseline, the Gal-9 expression percentage was higher in the group with severe disease than in mild or moderate groups. After treatment, the Gal-9 expression in CD3+, CD4+, CD8+ and CD4-CD8− cell subsets decreased, as well as Gal-9 mean fluorescence intensity in CD3+, CD4+ and CD8+ T cells. Similarly, plasma Gal-9 levels were lower at week 12 than at baseline. Good responders showed significantly lower Gal-9 expression on CD3+ and CD4+ T cell subsets and lower plasma Gal-9 levels than poor responders. Gal-9 expression positively correlates with disease activity in RA patients. Gal-9 can be regarded as a new biomarker for evaluating RA activity and therapeutic effect, including TAC.