Measurement of Lipoprotein-Associated Phospholipase A2 by Use of 3 Different Methods: Exploration of Discordance between ELISA and Activity Assays.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA2 have been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA2 measured by the standard ELISA-based immunoassays and the activity of this enzyme, leading to substantial discordance in risk categorization depending on assay format. METHODS: We developed 2 LC-MS/MS-based assays to quantify serum Lp-PLA2 activity (multiple reaction monitoring detection of product) and concentration [stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity], and we investigated their correlation to commercially offered colorimetric activity and immunometric concentrations assays. Associations between Lp-PLA2 and lipoproteins and the effect of selected detergents in liberating Lp-PLA2 were evaluated by use of immunoprecipitation and Western blot analyses. RESULTS: Serum Lp-PLA2 concentrations measured by quantitative SISCAPA-mass spectrometry were substantially higher than concentrations typically measured by immunoassay and showed an improved agreement with Lp-PLA2 activity. With detergents, liberation of Lp-PLA2 from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements. CONCLUSIONS: Quantitative analysis of Lp-PLA2 concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA2 concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA2 by immunoassay appears to be strongly inhibited by interaction of Lp-PLA2 with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA2 concentration (by SISCAPA) and activity (by direct product detection).

Severe dermatomyositis triggered by interferon beta-1a therapy and associated with enhanced type I interferon signaling.

BACKGROUND: Type I interferons (IFNs) are common therapeutics for several diseases, including viral infections and multiple sclerosis (MS). Although numerous studies have implicated type I INFs with the production of autoantibodies and the development of certain autoimmune disorders, interferon beta has not previously been described in association with dermatomyositis, to our knowledge. Previous microarray studies of muscle biopsy specimens from patients with dermatomyositis disclosed a type I IFN-induced gene expression profile. The central role of plasmacytoid dendritic cell precursors, together with increased type I IFN production, suggests a pivotal role for type I IFNs in dermatomyositis. We report a case of dermatomyositis exacerbated or induced by interferon beta therapy for MS and provide evidence that demonstrates enhanced type I IFN signaling in this patient. OBSERVATIONS: We observed new-onset dermatomyositis in a 57-year-old patient treated with interferon beta for MS. His symptoms were exacerbated temporally by interferon beta injections. Immunohistochemical staining of skin biopsy specimens for myxovirus-resistance protein A (a surrogate marker for cutaneous type I IFN signaling) showed increased staining that correlated temporally with interferon beta treatment and subsequent disease activity. In vitro treatment with interferon beta of peripheral blood mononuclear cells isolated from our patient revealed enhanced type I IFN signaling assessed by interferon-induced gene expression profiles. CONCLUSIONS: To our knowledge, this is the first description of dermatomyositis exacerbated or induced by interferon beta treatment. Our results demonstrate enhanced type I IFN signaling following interferon beta treatment in our patient with dermatomyositis.

Skin-infiltrating monocytes/macrophages migrate to draining lymph nodes and produce IL-10 after contact sensitizer exposure to UV-irradiated skin.

Low-dose UVB exposure induces antigen-specific unresponsiveness to antigen(s) introduced through UV-irradiated skin (tolerance). Analysis of cytokine expression in murine draining lymph nodes (DLNs) revealed that IL-12p40 mRNA and protein expression as well as IL-12p70 protein were upregulated after application of the contact sensitizer 2,4 dinitro-1-fluorobenzene (DNFB) to normal skin. The cellular source of IL-12p40 mRNA was CD11c+ cells. By contrast, following DNFB application to UV-irradiated skin (UV+DNFB), IL-12p40 mRNA was not upregulated, and DLN IL-12p40 and p70 proteins were reduced. UVB irradiation alone did not upregulate IL-10 mRNA, but UV+DNFB upregulated IL-10 mRNA as early as 3-6 hours after DNFB application, immediately preceding a decrease of IL-12p40 mRNA from the level induced by UVB. The infiltration of F4/80+ cells into UV-irradiated skin was followed by a rapid and remarkable increase of F4/80+CD11c(-) cells in DLN 3 hours following DNFB application. FITC/DNFB skin painting and subsequent enzyme-linked immunospot assay demonstrated that flow-sorted FITC+F4/80+CD11c(-) cells from the DLN produce IL-10. Thus, monocytes/macrophages that infiltrated into the skin following UVB exposure migrate to the DLN triggered by contact sensitizers. Production of IL-10 by migrating macrophages, in conjunction with IL-12 inhibition in the DLN, likely reflects a role as mobile suppressive mediators for locally induced UV tolerance.

Determination of in vivo dose response and allergen-specific T cells in subjects contact-sensitized to squaric acid dibutyl ester.

BACKGROUND: Squaric acid dibutyl ester (SADBE) is a known contact sensitizer, but dose-response data are not defined. OBJECTIVE: To determine the relationship between sensitization dose and contact hypersensitivity (CHS) response to SADBE in human volunteers. The study also aimed to investigate whether SADBE-reactive blood T cells could be detected using ex vivo mature dendritic cells (DCs) as antigen-presenting cells. METHOD: Forty healthy volunteers were sensitized to either 12.5, 25, 50, or 250 microg of SADBE in a 48 microL volume. This was followed by elicitation 2 weeks later with five doses (0, 0.2, 2, 20, and 200 microg in 20 microL). An additional 10 subjects received the elicitation doses without prior sensitization. Blood samples obtained after sensitization were purified into T cells and mature DCs. RESULTS: A direct relationship between sensitization dose and in vivo CHS response was observed. The SADBE dose that effectively sensitized 50% of the population (ED50) was 22 microg/cm2. Significant SADBE-specific T-cell proliferation in vitro was not observed 2 weeks after sensitization but became evident after elicitation. CONCLUSION: This study establishes the in vivo dose-response characteristics of immune reactivity to SADBE and antigen-specific T-cell reactivity.