Background: Tumor necrosis factor-α (TNFα) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFα, including those in healthy controls, cannot be measured by existing methods. Methods: We developed a sensitive immuno-PCR assay for the detection of TNFα in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFα sandwiched by antibodies was detected by amplification of the DNA label using PCR. Results: The limit of detection of the assay was 0.001 ng/L, an ∼5 × 104-fold improvement compared with a conventional ELISA. The mean serum TNFα concentration (± SD) in healthy donors was 0.021 ± 0.044 ng/L in men (n = 29) and 0.033 ± 0.065 ng/L in women (n = 25). Conclusion: This method may be useful for analyzing the significance of TNFα concentration in various diseases.

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The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI-TOF MS as an adduct with Phos-tag, a phosphate-capture zinc complex. Various α-hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC-MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4-hydroxysphingenine (dehydrophytosphingosine)-1-phosphate by MALDI-TOF MS with Phos-tag. From these results, lipid X was determined to be phytoceramide-1-phosphate (PC1P) with an α-hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants.

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Background Sphingosine-1-phosphate receptor 3 (S1P 3 ) is one of five receptors for sphingosine-1-phosphate (S1P). S1P/S1P 3 signaling is involved in numerous physiological and pathological processes including bone metabolism, sepsis, cancer, and immunity. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) are activated by several factors and promote abundant proinflammatory cytokine production and bone destruction. The aim of this study was to investigate whether S1P 3 is associated with the development of autoimmune arthritis and the pathogenic function of FLSs. Methods Wild-type (WT) and S1P 3 knockout (S1P 3 -KO) collagen-induced arthritis (CIA) mice were evaluated with respect to clinical and histological disease severity, along with the levels of anti-collagen antibodies and expression of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6). S1P 3 expression in the synovium was analyzed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining. FLSs isolated from CIA mice were activated with TNFα and S1P 3 expression was analyzed by real-time RT-PCR. The role of S1P/S1P 3 signaling in activated and non-activated FLSs was investigated by measuring cell proliferation and cyto/chemokine production by real-time RT-PCR and/or enzyme-linked immunosorbent assay. Results Clinical and histological scores, and synovial IL-6 expression were significantly lower in S1P 3 -KO mice with CIA than in WT mice. Arthritic synovia had higher S1P 3 expression than intact synovia and FLSs in arthritic joints expressed S1P 3 in vivo . Primary cultured FLSs produced IL-6 in a time-dependent manner in response to S1P stimulation and exhibited higher levels of S1P 3 expression after activation with TNFα. S1P 3 -induced production of IL-6 and MMP-3 was increased in FLSs pre-activated with TNFα. Conclusion In this study, we demonstrated that S1P 3 expression is associated with the development of autoimmune arthritis via inflammation-induced increases in S1P/S1P 3 signaling that increase production of IL-6 in FLSs. Inhibition of S1P/S1P 3 signaling could open the door to the development of new therapies for RA.