A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.     

The diabetogenic, insulin-specific CD8 T cell response primed in the experimental autoimmune diabetes model in RIP-B7.1 mice

Type 1 diabetes mellitus can result from the specific destruction of pancreatic beta cells by autoreactive T cells. As shown here, experimental autoimmune diabetes (EAD) is efficiently induced in RIP-B7.1 mice by preproinsulin (ppins)-encoding DNA vaccines. EAD develops in RIP-B7.1 mice within 3-4 wk after a single immunization with ppins-encoding plasmid DNA. RIP-B7.1 mice develop insulitis, insulin deficiency and hyperglycemia after vaccination with plasmids encoding murine ppins-I or murine ppins-II or human hu-ppins. EAD induction critically depends on CD8 T cells and is independent of CD4 T cells. To be diabetogenic, ppins-specific CD8 T cells had to express IFN-gamma. Neither expression of perforin nor signaling through the type I IFN receptor is an essential component of this pathogenic CD8 T cell phenotype. Using plasmids encoding truncated ppins variants, we show that EAD is only induced by DNA vaccines encoding the insulin A-chain. Diabetogenic CD8 T cells specifically recognize the Kb-restricted A12-21 epitope of the insulin A-chain. The RIP-B7.1 model hence represents an attractive model for the characterization of cellular and molecular events involved in the CD8 T cell-mediated immune pathogenesis of diabetes.     

Brief Report: First identification of H4 histamine receptor in healthy salivary glands and in focal sialadenitis in Sjögren's syndrome

Objective

The conventional H₁ and H₂ histamine receptors have >10,000‐fold lower avidity for histamine than H₄ histamine receptor, which has been implicated in autoimmune diseases. This study was undertaken to compare H₄ histamine receptor levels in the salivary glands (SGs) of healthy controls with those in the SGs of patients with primary Sjögren's syndrome (SS).

Methods

H₄ histamine receptor messenger RNA (mRNA) was analyzed using real‐time quantitative polymerase chain reaction, and the receptor protein was examined using immunostaining. Effects of the H₄ histamine receptor agonist ST‐1006 on cytokine synthesis by human SG (HSG) cells were analyzed using xMAP technology and enzyme‐linked immunosorbent assay.

Results

Healthy SGs contained H₄ histamine receptor mRNA. The receptor protein was localized to the acinar and ductal epithelial cells. H₄ histamine receptor agonist stimulated HSG cells to produce the cytokines interleukin‐8 and vascular endothelial growth factor. SS patients had low H₄ histamine receptor levels.

Conclusion

H₁ and H₂ histamine receptor antagonists are not effective in the treatment of autoimmune diseases. However, such antagonists do not affect the newly discovered H₄ histamine receptor. Dendritic cells and lymphocytes are nonprofessional histamine‐producing cells, which produce histamine at 100–1,000‐fold lower rates than mast cells do. Saliva contains only 0.31–12.4 ng/ml histamine, which is too low to stimulate H₁ or H₂ histamine receptor, but stimulates H₄ histamine receptor half maximally. Our findings show that H₄ histamine receptor is strongly expressed in tubuloacinar SG cells, which emphasizes the role of these cells in the pathogenesis of SS.