PubMed · 2940998
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H J Gurland, M J Lysaght, W Samtleben. 1986. Immunomodulation: clinical aspects.. https://doi.org/10.1111/j.1525-1594.1986.tb02530.x
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Antigen-driven immune responses are modulated by immune complexes (ICs), in part through their ability to inhibit IFN-gamma-dependent MHC Class II expression. We have demonstrated previously that ICs dramatically inhibit IFN-gamma-induced activation of human monocytes through the suppression of the JAK/STAT signaling pathway. In the current study, we further explore the mechanisms by which ICs regulate IFN-gamma activation of human monocytes. Consistent with previous studies in monocytes pretreated with ICs, there was a reduction in steady-state levels of RNA by real-time RT-PCR of the IFN-inducible protein 10 gene as well as the FcgammaRI gene. Pull-down assays confirm that IC pretreatment inhibits IFN-gamma-induced STAT1 phosphorylation without affecting the ability of STAT1 to bind to the STAT1-binding domain of the IFN-gamma receptor. In addition, the inhibitory function of ICs was reduced when cells from the FcR common gamma-chain knockout mice were used, supporting the role of the FcgammaRI in this inhibitory pathway. It is unexpected that ICs also require the phosphatase Src homology-2-containing tyrosine phosphatase 1 (SHP-1) to inhibit IFN-gamma induction, as demonstrated by studies with cells from the SHP-1 knockout (motheaten) mice. These data suggest a mechanism of IC-mediated inhibition of IFN-gamma signaling, which requires the ITAM-containing FcgammaRI, as well as the ITIM-dependent phosphatase SHP-1, ultimately resulting in the suppression of STAT1 phosphorylation.
In order to increase the sensitivity of liquid chromatography/mass spectrometry (LC/MS) assays of recombinant proteins for pharmacokinetics studies, we have developed an immuno-mass spectrometry assay for EPI-hNE4, a 6237 Da protein currently developed for respiratory distress syndromes. After immunocapture of the analyte in human plasma with magnetic beads coated with anti-EPI-hNE4 antibodies, the intact protein was eluted and separated in reversed-phase LC and then analysed by tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) mode. The problem of analytical interference due to endogenous binding antibodies was addressed by successive steps of acidification and neutralisation before immunocapture. Furthermore, potential variations in the recovery of analyte during sample extraction were compensated for by addition of an internal standard recognised by the antibodies. The precision of the assay remained therefore below 15%. A significant increase in assay sensitivity was achieved since the extraction step allowed sample concentration and removal of matrix components interfering with the electrospray ionisation process. Using 0.4 mL of plasma, a limit of quantification at 0.5 ng/mL (80 pM) was reached, which represents a 10-fold improvement in sensitivity over our previous work using sample precipitation. This technique was able to monitor EPI-hNE4 kinetics in the plasma of human subjects for 36 h after an intravenous administration of 0.125 mg/kg.
Detection of multiple surface antigens expressed on living cell is an important step for cell processing and clinical diagnosis. Here we describe the preparation of antibody arrays that allow parallel detection of multiple surface antigens through affinity binding of living cells. An antibody array was fabricated by photo-assisted patterning of an alkanethiol monolayer formed on a gold-coated glass plate and subsequent immobilization of antibodies specific for cell surface antigens in an array format. We demonstrate here that rapid phenotyping can be performed on the array for both adhesion-dependent and non-dependent cells by direct cell binding assays. The density of bound cells on each antibody spot was in accordance with their contents in an original suspension. This result suggests the feasibility of the array-based method for quantitative assessment of multiple antigen expression. These findings will serve to extend the range of fundamental and clinical applications of antibody arrays.