Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts.

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK-as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-ß (TGF-ß) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-ß immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-ß nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-ß receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-ß are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts.

Interleukin-1 receptor-mediated inflammation impairs the heat shock response of human mesothelial cells.

Bioincompatibility of peritoneal dialysis fluids (PDF) limits their use in renal replacement therapy. PDF exposure harms mesothelial cells but induces heat shock proteins (HSP), which are essential for repair and cytoprotection. We searched for cellular pathways that impair the heat shock response in mesothelial cells after PDF-exposure. In a dose-response experiment, increasing PDF-exposure times resulted in rapidly increasing mesothelial cell damage but decreasing HSP expression, confirming impaired heat shock response. Using proteomics and bioinformatics, simultaneously activated apoptosis-related and inflammation-related pathways were identified as candidate mechanisms. Testing the role of sterile inflammation, addition of necrotic cell material to mesothelial cells increased, whereas addition of the interleukin-1 receptor (IL-1R) antagonist anakinra to PDF decreased release of inflammatory cytokines. Addition of anakinra during PDF exposure resulted in cytoprotection and increased chaperone expression. Thus, activation of the IL-1R plays a pivotal role in impairment of the heat shock response of mesothelial cells to PDF. Danger signals from injured cells lead to an elevated level of cytokine release associated with sterile inflammation, which reduces expression of HSP and other cytoprotective chaperones and exacerbates PDF damage. Blocking the IL-1R pathway might be useful in limiting damage during peritoneal dialysis.

The Extracorporeal Proteome-The Significance of Selective Protein Removal During Dialysis Therapy.

Dialysis as renal replacement therapy aims excess water and waste solutes from the uremic patient while retaining proteins in the plasma. Irrespective of the dialysis modality, hemodialysis (HD) or peritoneal dialysis (PD), the amount and composition of proteins that are removed are important determinants of the biocompatibility of the therapy. Although hemodialysis membranes would ideally be biologically inert filtration tubes, they are known to adsorb proteins. The part of the plasma proteome that is thereby removed during every dialysis session may be regarded as the extracorporeal proteome, which has to be kept in balance with the plasma proteome, regarding the individual proteins' biological roles and activation states. In a recent study, Ronci et al. (Proteomics Clin. Appl. 2018, e1700140) comprehensively compare two hemodialyzer membrane materials by shotgun LC-MS proteomic analysis of adsorbed proteins and ultrafiltrates from four HD patients. While pathway analysis is an attractive tool to compare different proteomes on an abstract level, some challenges remain regarding the adaptation for such tools for special proteomes and the interpretation of relative changes compared absolute changes regarding their biological importance in dialysis techniques. In summary, selective protein removal may represent a yet unexploited therapeutic opportunity if the "right" proteins are removed from the blood.

HSP induction in mesothelial cells by peritoneal dialysis fluid depends on biocompatibility test system.

BACKGROUND: We have previously shown that exposure of mesothelial cells (MC) to peritoneal dialysis fluids (PDF) not only caused toxic injury, but also induced cytoprotective heat shock proteins (HSP). This study was performed in order to compare HSP expression in MC upon PDF exposure in three currently used biocompatibility test systems. METHODS: Omentum-derived human peritoneal MC underwent 3 modalities of exposure to heat- or filter-sterilized PDF: (A) pure PDF for 60 minutes followed by a recovery-period in pure culture medium for 24 hours; (B) 1:1 mixture of PDF and culture medium for 24 hours or (C) pure PDF for 60 minutes followed by a recovery-period in a 1:1 mixture of PDF and culture medium for 24 hours. Biocompatibility was assessed by LDH-release into the supernatant and HSP-72 expression in MC lysates. RESULTS: Short-term exposure of MC to pure PDF (Modality A) resulted in concordant LDH release and upregulation of HSP-72, regardless of heat or filter sterilization. In contrast, both test systems that exposed MC to heat-sterilized PDF during the recovery period (Modalities B and C) resulted in severe cellular lethality but low HSP-72 expression. CONCLUSIONS: This study clearly shows that HSP expression in MC upon PDF exposure depends on the biocompatibility test system. The presence of heat-sterilized PDF during recovery resulted in significant downregulation of Hsp-72 despite severe cell injury. Therefore, Hsp-72 expression reflects adequate cellular stress responses rather than PDF cytotoxicity.