Expression of proteasomal immunosubunit beta1i is dysregulated in inflammatory infiltrates of minor salivary glands in Sjogren's syndrome.

OBJECTIVE: Minor salivary gland specimens were analyzed to investigate dysregulation of the proteasome system in patients with Sjögren's syndrome (SS) and patients with sicca syndrome. METHODS: Labial biopsy specimens from 17 patients with SS and 11 patients with non-autoimmunesicca syndrome were analyzed by immunohistochemistry for expression of the inducible proteasomal subunits ss1i, ss2i, and ss5i. The infiltrating subsets of lymphocytes were characterized using immunofluorescence stainings against the cell-surface markers CD20 and CD27. Two-dimensional electrophoresis and immunoblotting were used for detection of the proteasomal subunits ss1 and ss1i in peripheral blood monocyte cells. Gene expression of the constitutive subunits ss1, ss2, and ss5 and the corresponding inducible subunits ss1i, ss2i, and ss5i was further investigated at the mRNA level in small lip biopsies using real-time polymerase chain reaction. RESULTS: The expression of ss1i in infiltrating and peripheral immune cells was altered in patients with SS compared to patients with non-autoimmune sicca syndrome and healthy controls. No significant differences were found in ss2i and ss5i expression between the same groups in small lip biopsies. Chisholm-Mason grade and ss1i expression were found to be inversely correlated (Spearman r = -0.461, p = 0.014). The phenotype and distribution of the lymphocytic infiltrate showed no differences between patients with primary and secondary SS regardless of ss1i expression. CONCLUSION: The proteasomal ss1i subunit is dysregulated in peripheral white blood cells and in inflammatory infiltrates of minor salivary glands in patients with SS.

Autoantibodies to asialoglycoprotein receptor (ASGPR) measured by a novel ELISA--revival of a disease-activity marker in autoimmune hepatitis.

BACKGROUND: The liver-specific ASGPR is an autoantigen in autoimmune hepatitis (AIH) patients. Anti-ASGPR antibody correlates with disease activity, however, only in-house assays have been reported so far. METHODS: Rabbit ASGPR was purified by affinity chromatography on galactose-Sepharose and used for standardised detection of anti-ASGPR by ELISA. Anti-ASGPR IgG was measured in sera from 45 patients with AIH, PBC (n=43), alcoholic liver disease (n=13), HBV infection (n=35), HCV infection (n=53), and 118 blood donors. Anti-ASGPR was correlated with biochemical parameters of disease activity in 22 AIH patients with consecutive samples. RESULTS: Twenty-one of 30 untreated (70%) and five of 15 treated AIH patients (30%) showed elevated anti-ASGPR at first presentation. Only one blood donor demonstrated anti-ASGPR. ALD and PBC patients were all negative. ROC curve analysis of AIH and disease-control patients revealed a sensitivity of 77.8% and a specificity of 99.4%. Three (8.6%) of 35 HBV and 7 (13.2%) of 53 HCV patients demonstrated elevated anti-ASGPR. In AIH patients, anti-ASPGR correlated with liver-transaminases levels. In 22 follow-up patients, elevation of anti-ASPGR preceded liver-transaminases increase. CONCLUSIONS: The novel anti-ASGPR ELISA is a readily available and specific diagnostic tool for anti-ASGPR detection in AIH. Quantification of anti-ASGPR is helpful in monitoring disease activity.

Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1.

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1+ cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies.

Anti-20S proteasome autoantibodies inhibit proteasome stimulation by proteasome activator PA28.

OBJECTIVE: The ubiquitin-proteasome system plays a central role in cellular homeostasis as well as in regulation of the inflammatory and stress responses. However, the occurrence of autoantibodies against 20S proteasome has, to date, been considered to be a non-specific epiphenomenon in patients with autoimmune disorders. This study was undertaken to analyze the properties of antiproteasome antibodies by investigating their influence on the proteolytic activity of the proteasome complex. METHODS: The 20S proteasome, with or without addition of the proteasome activator (PA28), was preincubated with affinity-purified human antiproteasome antibodies. Proteolytic activity was estimated using fluorogenic peptides as substrate. RESULTS: The baseline proteolytic properties of the 20S proteasome core complex were not influenced by the autoantibodies in vitro. In contrast, all human antiproteasome antibodies analyzed efficiently blocked the enhanced proteasomal substrate cleavage provided by PA28. A similar influence of proteasome activation was observed upon preincubation with affinity-purified sheep polyclonal or mouse monoclonal antiproteasome antibody, whereas human immunoglobulin controls exhibited no effect. CONCLUSION: Autoantibodies against 20S proteasomes are able to block proteasome activation by PA28, binding to their native antigen in vitro. Antibody targeting of the interaction between 20S proteasome and PA28 represents a novel mechanism of proteasome inhibition.