high-throughput-flow-cytometry-cell-based-assay-to-detect-antibodies

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Westmead Millennium Institute for Medical Research

Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces-stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-beta1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis.

Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line.

Fibroblast activation protein (FAP) is the member of Dipeptidyl Peptidase IV (DPIV) gene family that is most similar to DPIV. Four members of this family, DPIV, FAP, DP8 and DP9 possess a rare catalytic activity, hydrolysis of a prolyl bond two residues from the substrate N terminus. Crystal structures show that the soluble form of FAP comprises two domains, an alpha/beta-hydrolase domain and an 8-blade beta-propeller domain. The interface between these two domains forms the catalytic pocket, and an opening for substrate access to the internal active site. The FAP homodimer is structurally very similar to DPIV but FAP glycoprotein expression is largely confined to mesenchymal cells in diseased and damaged tissue, notably the tissue remodelling region in chronically injured liver. FAP peptide substrates include denatured collagen and alpha2-antiplasmin. The functional roles of FAP in tumors and fibrotic tissue are not fully understood. This review places FAP in the context of chronic liver injury pathogenesis.

Fibroblast activation protein and chronic liver disease.

Autoreactive T cells play a pivotal role in the pathogenesis of autoimmune kidney disease. T cell vaccination (TCV) may limit autoimmune disease and induce CD8+ regulatory T cells (Tregs). We used Heymann nephritis (HN), a rat model of human membranous nephritis, to study the effects of TCV on autoimmune kidney disease. We harvested CD4+ T cells from renal tubular antigen (Fx1A) -immunized rats and activated these cells in vitro to express the MHC Class Ib molecule Qa-1. Vaccination of Lewis rats with these autoreactive Fx1A-induced T cells protected against HN, whereas control-primed T cells did not. Rats that underwent TCV had lower levels of proteinuria and serum creatinine and significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates. Furthermore, these rats expressed less IFN-γ and IL-6 in splenocytes, whereas the numbers of Tregs and the expression of Foxp3 were unchanged. In vitro cytotoxicity assays showed CD8+ T cell-mediated elimination of Qa-1-expressing CD4+ T cells. In vivo, TCV abrogated the increase in Qa-1-expressing CXCR5+ TFH cells observed in HN compared with controls. Taken together, these results suggest that TCV protects against autoimmune kidney disease by targeting Qa-1-expressing autoreactive CD4+ cells.

Xin Maggie Wang

Flow Cytometry Centre

Westmead Millennium Institute for Medical Research

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Xin Maggie Wang

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Westmead Millennium Institute for Medical Research

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Flow Cytometry Centre