EBNA1 IgM-Based Discrimination Between Rheumatoid Arthritis Patients, Systemic Lupus Erythematosus Patients and Healthy Controls

Epstein–Barr Virus (EBV) has been associated with development of rheumatic connective tissue diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in genetically susceptible individuals. Diagnosis of RA and SLE relies on clinical criteria in combination with the presence of characteristic autoantibodies. In addition, antibodies to several EBV antigens have been shown to be elevated in patients with these diseases compared to healthy controls (HC). Here, we elaborated improved enzyme-linked immunosorbent assays for antibodies (IgM, IgA, IgG) to the EBV proteins Epstein-Barr Virus nuclear antigen (EBNA)1 and early antigen diffuse (EAD) in order to determine their potential diagnostic role. We showed that especially EBNA1 IgM distinguished RA from SLE and HCs and also distinguished SLE from HCs. EBNA1 IgA was almost as effective in differentiating RA from SLE and HC, while EAD IgG and IgA were able to discern SLE patients from RA patients and HCs. Collectively, these findings illustrate the potential diagnostic use of antibodies to EBV proteins to diagnose RA and to differentiate SLE from RA

Connecting sialic acid expression to cancer cell characteristics : Novel tools for detection, imaging, and analysis

Sialic acid (SA) plays a crucial role in many biological processes. Cell surface SA expression is usually analyzed with antibodies or lectins; however, they are costly and with poor stability. We have used a molecular imprinting technique to synthesize an alternative SA receptor – SA molecularly imprinted polymers(SA-MIPs) with an embedded fluorophore for fluorescent detection of theSA-MIPs. The binding behavior and specificity of SA-MIPs were verified by using lectins and SA conjugates on cancer cell lines, showing that SA-MIPs can be used as an effective tool for SA expression analysis of cancer cells. Digital holographic cytometry (DHC) is a non-phototoxic quantitative phase imaging technique that facilitates the monitoring of living cells over time. We have demonstrated the potential of DHC by mapping cellular parameters, such as cell number, area, thickness, and volume. In addition, cellular parameters possibly depending on sialylation, were evaluated using DHC. Furthermore, the uptake over time of SA-MIPs by macrophages was investigated for any inflammatory and/or cytotoxic responses when administered to phagocytosing cells. Our results indicate that SA-MIPs caused low induction and sparse secretion of inflammatory cytokines, and that reduced cell proliferation was not due to cytotoxicity, but to attenuated cell cycles. These results suggest that SA-MIPs will contribute to the further understanding of cancer cell behavior and can be an asset for in vivo studies.Incorrect ISBN in publication: 978-91-7877-326-1 (pdf)</p

Digital Holographic Microscopy : Macrophage Uptake of Nanoprobes

Digital holographic cytometry (DHC) is a state-of-the-art quantitative phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped with semi-automated image segmentation and analysis software packages for assessing cell behavior are commercially available. In this study we investigate the possible uptake of nanoprobes in macrophages in vitro over time

Digital Holographic Microscopy : Macrophage Uptake of Nanoprobes

Digital holographic cytometry (DHC) is a state-of-the-art quantitative phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped with semi-automated image segmentation and analysis software packages for assessing cell behavior are commercially available. In this study we investigate the possible uptake of nanoprobes in macrophages in vitro over time

Digital Holographic Microscopy : Macrophage Uptake of Nanoprobes

Digital holographic cytometry (DHC) is a state-of-the-art quantitative phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped with semi-automated image segmentation and analysis software packages for assessing cell behavior are commercially available. In this study we investigate the possible uptake of nanoprobes in macrophages in vitro over time

Molecularly Imprinted Polymers Exhibit Low Cytotoxic and Inflammatory Properties in Macrophages In Vitro

Molecularly imprinted polymers (MIPs) against sialic acid (SA) have been developed as a detection tool to target cancer cells. Before proceeding to in vivo studies, a better knowledge of the overall effects of MIPs on the innate immune system is needed. The aim of this study thus was to exemplarily assess whether SA-MIPs lead to inflammatory and/or cytotoxic responses when administered to phagocytosing cells in the innate immune system. The response of monocytic/macrophage cell lines to two different reference particles, Alhydrogel and PLGA, was compared to their response to SA-MIPs. In vitro culture showed a cellular association of SA-MIPs and Alhydrogel, as analyzed by flow cytometry. The reference particle Alhydrogel induced secretion of IL-1β from the monocytic cell line THP-1, whereas almost no secretion was provoked for SA-MIPs. A reduced number of both THP-1 and RAW 264.7 cells were observed after incubation with SA-MIPs and this was not caused by cytotoxicity. Digital holographic cytometry showed that SA-MIP treatment affected cell division, with much fewer cells dividing. Thus, the reduced number of cells after SA-MIP treatment was not linked to SA-MIPs cytotoxicity. In conclusion, SA-MIPs have a low degree of inflammatory properties, are not cytotoxic, and can be applicable for future in vivo studies

Molecularly imprinted polymers in biological applications.

Molecularly imprinted polymers (MIPs) are currently widely used and further developed for biological applications. The MIP synthesis procedure is a key process, and a wide variety of protocols exist. The templates that are used for imprinting vary from the smallest glycosylated glycan structures or even amino acids to whole proteins or bacteria. The low cost, quick preparation, stability and reproducibility have been highlighted as advantages of MIPs. The biological applications utilizing MIPs discussed here include enzyme-linked assays, sensors, in vivo applications, drug delivery, cancer diagnostics and more. Indeed, there are numerous examples of how MIPs can be used as recognition elements similar to natural antibodie

Efficient evaluation of humoral immune responses by the use of serum pools

Background: Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. Methods: We created serum pools from four different patient groups (systemic lupus erythematosus n = 85, rheumatoid arthritis n = 77, Sjögren's syndrome n = 91, systemic sclerosis n = 66) and one healthy control group (n = 67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. Results: The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. Conclusion: We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors

Fluorescent Molecularly Imprinted Polymer Layers against Sialic Acid on Silica-Coated Polystyrene Cores-Assessment of the Binding Behavior to Cancer Cells.

Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells