Anti-SAA1 monoclonal antibody, clone TBB26

Amyloid A (AA) amyloidosis is a systemic protein misfolding disease affecting humans and other vertebrates. While the protein precursor in humans and mice is the acute-phase reactant serum amyloid A (SAA) 1.1, the deposited fibrils consist mainly of C-terminally truncated SAA fragments, termed AA proteins. For yet unknown reasons, phenotypic variations in the AA amyloid distribution pattern are clearly associated with specific AA proteins. Here we describe a bacterial expression system and chromatographic strategies to obtain significant amounts of C-terminally truncated fragments of murine SAA1.1 that correspond in truncation position to relevant pathological AA proteins found in humans. This enables us to investigate systematically structural features of derived fibrils. All fragments form fibrils under nearly physiological conditions that show similar morphological appearance and amyloid-like properties as evident from amyloid-specific dye binding, transmission electron microscopy and infrared spectroscopy. However, infrared spectroscopy suggests variations in the structural organization of the amyloid fibrils that might be derived from a modulating role of the C-terminus for the fibril structure. These results provide insights, which can help to get a better understanding of the molecular mechanisms underlying the different clinical phenotypes of AA amyloidosis.

Background Intervertebral disc degeneration (IVDD) is a well-known cause of lower back pain, which is induced by multiple factors including increased apoptosis and decreased survival of nucleus pulposus cells. In this study, we evaluate the effect and potential mechanism of miR-660 on the nucleus pulposus cells apoptosis induced by TNF-alpha. Methods First, we collected tissue of nucleus pulposus from IVDD and healthy controls. General characteristic of the IVDD and healthy control was also collected. And, we also collected nucleus pulposus cells that stimulated by TNF-alpha or control. miRNA microarray was performed to identify the differentially expressed miRNAs. Apoptosis rate and miR-660 relative expression was measured after stimulated with different concentration of TNF-alpha to identify the optimal concentration of TNF-alpha. Second, we successfully constructed antigomiR-660 to block the miR-660 expression in nucleus pulposus cells and then stimulated with TNF-alpha (100 ng/ml, 12 h). The apoptosis rates and relative protein expression were then measured again. The target association between miR-660 and SAA1 was confirmed by dual-luciferase reporter. Results There was no significant difference between the age (IVDD: 39 +/- 10 years, healthy controls: 36 +/- 7 years), BMI and sex between IVDD and healthy controls. Microarray analysis found that miR-660 was significantly up-regulated in IVDD and TNF-alpha treated groups, which was further identified by PCR. We found that the rate of apoptosis and miR-660 expression increased with TNF-alpha concentration increased. Finally, TNF-a with 100 ng/ml was used for further experiment. Compared with TNF-alpha group, TNF-alpha + antigomiR-660 could significantly down-regulated the apoptosis rate and relative protein (c-Caspase3 and c-Caspase7). Dual-luciferase reporter revealed that miR-660 could directly binding to the SAA1 at 80-87 sites. Compared with TNF-alpha alone group, TNF-alpha + antigomiR-660 significantly up-regulated the SAA1 expression (P < 0.05). Conclusion These results indicated that knockdown of miR-660 protected the nucleus pulposus from apoptosis that induced TNF-alpha via up-regulation of SAA1. Further studies should focus on the role of miR-660 in protecting IVDD in vivo.