Recombinant thrombomodulin attenuates hyper-inflammation and glycocalyx damage in a murine model of Streptococcus pneumoniae-induced sepsis.

PURPOSE: The anticoagulant agent recombinant thrombomodulin (rTM) activates protein C to prevent excessive coagulation and also possibly regulates hyper-inflammation via neutralization of high-mobility-group B1 (HMG-B1). The glycocalyx layer in endothelial cells also plays a pivotal role in preventing septic shock-associated hyperpermeability. The present study examined the effect of rTM in a murine model of Streptococcus pneumoniae-induced sepsis. METHODS: Male C57BL/6N mice were injected intratracheally via midline cervical incision with 2 × 107 CFU of S. pneumoniae (capsular subtype 19A). Control mice were sham-treated identically but injected with saline. rTM (10 mg/kg) was injected intraperitoneally 3 h after septic insult. Blood concentrations of soluble inflammatory mediators (interleukin [IL]-1ß, IL-6, IL-10, and tumor necrosis factor [TNF]-α) were determined using a microarray immunoassay. Serum concentrations of HMG-B1 and syndecan-1, as a parameter of glycocalyx damage, were determined by enzyme-linked immunosorbent assay. The glycocalyx was also evaluated with electron microscopy. The lungs were removed, and digested to cells, which were then stained with a mixture of fluorophore-conjugated antibodies. Anti-mouse primary antibodies included PE-Cy7-conjugated anti-CD31, AlexaFluor 700-conjugated anti-CD45, PerCP-Cy5.5-conjugated anti-CD326, APC-conjugated anti-TNF-α, PE-conjugated anti-IL-6, and PE-conjugated anti-IL-10. A total of 1 × 106 cells per sample were analyzed, and 2 × 105 events were recorded by flow cytometry, and parameters were compared with/without rTM treatment. RESULTS: The blood concentration of TNF-α was significantly reduced 24 h after intratracheal injection in S. pneumoniae-challenged mice treated with rTM (P = 0.016). Levels of IL-10 in the lung endothelium of rTM-treated S. pneumoniae-challenged mice increased significantly 12 h after intratracheal injection (P = 0.03). Intriguingly, serum HMGB-1 and syndecan-1 levels decreased significantly (P = 0.010 and 0.015, respectively) in rTM-treated mice 24 h after intratracheal injection of S. pneumoniae. Electron microscopy indicated that rTM treatment preserved the morphology of the glycocalyx layer in septic mice. CONCLUSIONS: These data suggest that rTM modulates local inflammation in the lung endothelium, thus diminishing systemic inflammation, i.e., hypercytokinemia. Furthermore, rTM treatment reduced serum syndecan-1 levels, thus preventing glycocalyx damage. The use of rTM to treat sepsis caused by bacterial pneumonia could therefore help prevent both excessive inflammation and glycocalyx injury in the lung endothelium.

Establishment of mouse gingival junctional epithelial cell line using a bioengineered tooth system.

Junctional epithelium (JE), one of the constituents of periodontal tissue, has several unique features to prevent bacterial infection. However, the molecular mechanisms of these cells remain to be completely elucidated because there has been no JE cell line to date. We have succeeded in isolating JE cells expressing green fluorescent protein (GFP) by using a bioengineered tooth technique in mice. The gene expressions of GFP-positive JE cells, isolated from around the erupted bioengineered teeth using flow cytometry, were analyzed by RNA sequencing. GFP-positive cells derived from the bioengineered tooth germs showed similar gene expression patterns to primary JE cells. The isolated GFP-positive JE cells were immortalized by transducing the simian virus 40 large T antigen using lentiviral vectors. The established GFP-positive JE cells maintained proliferative activity for more than 20 passages, and did not show cellular senescence as demonstrated by ß-galactosidase assay. These cells also expressed similar gene expression patterns to primary JE cells. The established cell lines may prove useful for future investigation of JE characteristics in vitro.

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110.

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell-cell communications and host-microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.

Visualization of junctional epithelial cell replacement by oral gingival epithelial cells over a life time and after gingivectomy.

Junctional epithelium (JE), which is derived from odontogenic epithelial cells immediately after eruption, is believed to be gradually replaced by oral gingival epithelium (OGE) over a lifetime. However, the detailed process of replacement remains unclear. The aim of the present study was to clarify the process of JE replacement by OGE cells using a green fluorescent protein (GFP)-positive tooth germ transplantation method. GFP-positive JE was partly replaced by OGE cells and completely replaced on day 200 after transplantation, whereas there was no difference in the expression of integrin ß4 (Itgb4) and laminin 5 (Lama5) between JE before and after replacement by OGE cells. Next, GFP-positive JE was partially resected. On day 14 after resection, the regenerated JE consisted of GFP-negative cells and also expressed both Itgb4 and Lama5. In addition, the gene expression profile of JE derived from odontogenic epithelium before gingivectomy was partly different from that of JE derived from OGE after gingivectomy. These results suggest that JE derived from the odontogenic epithelium is gradually replaced by OGE cells over time and JE derived from the odontogenic epithelium might have specific characteristics different to those of JE derived from OGE.

Simultaneous genotyping of indels and SNPs by mass spectroscopy.

Nucleotide insertion/deletion polymorphisms (indels) in ApoE gene were precisely genotyped using artificial ribonucleases and MALDI-TOF MS. The RNA fragments for MS analysis were prepared by treating RNA specimens with our artificial ribonucleases, which consist of LuCl(3) (molecular scissors) and oligonucleotides bearing two acridine groups (RNA-activator for site-selective scission). RNA scission by Lu(III) ion always occurred at the phosphodiester linkages in front of the two acridines, even when the RNA specimens involved consecutive cytidine sequences of different lengths. Thus, even complicated mixtures of these indel specimens were completely genotyped by using only one acridine-bearing oligonucleotide and by subjecting the reaction mixture to single MS measurement. Moreover, single nucleotide polymorphism (SNP) in the consecutive sequences could be genotyped simultaneously with the indels.

Site-selective RNA scission by PNA-Lu(III) hybrid system.

By using two PNA (PNA = peptide nucleic acid) additives, one-base gap structure was formed in RNA. Upon treatment of this gap-forming RNA substrate with Lu(III) ion, the phosphodiester linkage at 5'-side of the unpaired nucleotide was mainly cleaved. Promising feature of PNA as a sequence recognizing moiety of man-made RNA cutters was evidenced.