Evaluation of a Novel Immunochromatographic Device for Detecting Porphyromonas gingivalis in Patients with Periodontal Disease.

Porphyromonas gingivalis is the most pathogenic periodontal bacterium in the world. Recently, P. gingivalis has been considered responsible for dysbiosis during the development of periodontitis. This study aimed to evaluate a novel immunochromatographic device using monoclonal antibodies against P. gingivalis in subgingival plaques. A total of 72 patients with chronic periodontitis and 53 periodontally healthy volunteers underwent clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis and compared using real-time polymerase chain reaction (PCR). In the periodontitis group, a significant positive correlation was observed between the test device scores and the real-time PCR results. The specificity, positive predictive value, negative predictive value, and accuracy of the test device for P. gingivalis, as determined by real-time PCR, were 98%, 94%, 89%, and 90%, respectively. There were significant differences in bacterial counts by real-time PCR among the groups with different ranges of device scores. Additionally, there was a significant positive correlation between the device scores for P. gingivalis and periodontal parameters. These results suggest that this novel immunochromatographic device can be effectively used for rapid detection and semi-quantification of P. gingivalis in subgingival plaques.

The Ability of a Novel Trypsin-like Peptidase Activity Assay Kit to Detect Red-Complex Species.

The trypsin-like peptidase activity assay kit measures the trypsin-like protease produced by three red-complex species, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, causing periodontitis, and detects the presence of these bacteria in samples. The purpose of this study was to investigate the relationship between the detection of TLPs by a novel TLP-AA, ADCHECK and the detection of red-complex pathogens by real-time PCR using tongue swabs from patients with periodontitis. The detection limit of trypsin-like protease activity by ADCHECK was validated using the culture supernatants of two different Porphyromonas gingivalis bacterial strains. Real-time PCR was performed to determine the number of red-complex species in the tongue coatings of patients with periodontal disease. Trypsin-like protease activity in tongue-swab samples was scored using ADCHECK. ADCHECK successfully detected trypsin-like protease activity in 103 Porphyromonas gingivalis bacterial strains. The specificity, positive predictive value, negative predictive value, and accuracy of ADCHECK for the presence of red-complex pathogens determined by real-time PCR were 90%, 97%, 98%, and 92%, respectively. ADCHECK is an effective tool for the detection of red-complex pathogens.

Associations of cytokine levels in gingival crevicular fluid of mobile teeth with clinical improvement after initial periodontal treatment.

Studies suggest that analysis of gingival crevicular fluid (GCF) is useful for evaluating periodontal status. In this study, clinical variables related to tooth mobility, and multiple cytokine levels in proximate GCF, were measured at four time points during initial periodontal treatment: before treatment (baseline), after supragingival scaling, after occlusal adjustment, and after scaling and root planing (SRP); 20 teeth from 13 patients with periodontitis were included. Baseline interleukin (IL)-10 level in GCF was significantly higher around teeth that showed substantial improvement in periodontal epithelial surface area (PESA) after SRP than around teeth without PESA improvement. IL-3 and IL-16 levels in GCF at baseline were significantly higher around teeth with a periodontal inflamed surface area (PISA) of 0 mm2 after SRP than around teeth without PISA improvement. In addition, baseline IL-7, IL-11, and IL-12p40 levels in GCF were significantly lower around teeth with decreased mobility after occlusal adjustment than around teeth without decreased mobility. These results suggest that pre-treatment cytokine levels in GCF are useful in predicting the effects of initial periodontal treatment.

Identification of multiple amyloidogenic sequences in laminin-1.

Amyloid fibril formation is associated with several pathologies, including Alzheimer's disease, Parkinson's disease, type II diabetes, and prion diseases. Recently, a relationship between basement membrane components and amyloid deposits has been reported. The basement membrane protein, laminin, may be involved in amyloid-related diseases, since laminin is present in amyloid plaques in Alzheimer's disease and binds to amyloid precursor protein. Recently, we showed that peptide A208 (AASIKVAVSADR), the IKVAV-containing peptide, formed amyloid-like fibrils. We previously identified 60 cell adhesive sequences in laminin-1 using a total of 673 12-mer synthetic peptides. Here, we screened for additional amyloidogenic sequences among 60 cell adhesive peptides derived from laminin-1. We first examined amyloid-like fibril formation by the 60 active peptides with Congo red, a histological dye binding to many amyloid-like proteins. Thirteen peptides were stained with Congo red. Four of the 13 peptides promoted cell attachment and neurite outgrowth like the IKVAV-containing peptide. The four peptides also showed amyloid-like fibril formation in both X-ray diffraction and electron microscopic analyses. The amyloidogenic peptides contain consensus amino acid components, including both basic and acidic amino acids and Ser and Ile residues. These results indicate that at least five laminin-derived peptides can form amyloid-like fibrils. We conclude that the laminin-derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo, possibly when laminin-1 is degraded.

Identification of neurite outgrowth active sites on the laminin alpha4 chain G domain.

The laminin alpha4 chain is widely distributed in various mesodermal tissues, including the perineurium of peripheral nerves, dorsal root ganglion (DRG), skeletal muscle, and capillaries, and plays important roles in synaptic specialization at the neuromuscular junction and in microvascular formation. The C-terminal globular domain (G domain) of the laminin alpha4 chain was previously found to be critical for heparin binding and cell attachment activity. Here, we focused on neurite outgrowth activity of the laminin alpha4 chain G domain. We found that the recombinant alpha4 chain G domain protein (rec-alpha4G) promoted neurite outgrowth of rat pheochromocytoma PC12 cells. When 114 overlapping synthetic peptides that covered the entire G domain were tested for neurite outgrowth activity, nine peptides were active, but the 105 remaining peptides did not exhibit activity. Three of the nine active peptides, A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), and A4G107 (VIRDSNVVQLDV), strongly promoted neurite outgrowth of PC12 cells. A4G107 was found to form amyloid-like fibrils in Congo red, X-ray, and electron microscopy analyses. We also synthesized cyclic peptides to evaluate their conformational requirements. Cyclic peptide A4G82X (cyc-A4G82X;TLFLAHGRLVFX, where X is norleucine) significantly enhanced neurite outgrowth activity, but the rest of the cyclic peptides eliminated the activity. The A4G82 sequence is located on the loop region, suggesting that the activity of A4G82 is required for a loop conformation. These peptides also exhibited neurite outgrowth activity with dorsal root ganglion (DRG) explants and with DRG cells from E14.5 mouse embryos, indicating that they are active in both neuronal cell lines and native neuronal cells. Taken together, the data suggest that the peptides from the laminin alpha4 chain G domain promote neurite outgrowth activity via a specific conformation.

Multifunctional peptide fibrils for biomedical materials.

The Ile-Lys-Val-Ala-Val (IKVAV) containing peptide, A208 (AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108), was recently found to form amyloid-like fibrils. Fibril formation is critical for its biological activities, including promotion of cell adhesion and neurite outgrowth. In the present study, we designed multifunctional peptide fibrils using the A208 peptide and an Arg-Gly-Asp (RGD)-containing fibronectin active sequence for biomedical applications. The fibronectin active sequence GRGDS (FN) or a scrambled sequence RSGGD (SC) were conjugated to either A208 or to A208S (AASVVIAKSADR), a scrambled peptide of A208, with a glycine as a spacer. The FN-A208 and SC-A208 peptides formed a gel and were stained with Congo red similar to that of A208, but FN-A208S and SC-A208S did not form a gel. These results indicate that FN-A208 and SC-A208 form amyloid-like fibrils similar to A208. A208 and SC-A208 promoted cell attachment with filopodia formation, and this adhesion was inhibited by the IKVAV-containing peptide, but not by EDTA or a GRGDS peptide. FN-A208 promoted cell attachment with well-organized actin stress fibers, and this adhesion was partially inhibited by either EDTA, GRGDS, or IKVAV. These data suggest that A208 binds to only IKVAV receptor(s) while the FN-A208 interacts with both integrins and the IKVAV receptor(s). We conclude that multifunctional peptide fibrils can be designed by conjugation of active peptides on A208 and that this construct has potential to serve as a bioadhesive for tissue regeneration and engineering.

Syndecan binding sites in the laminin alpha1 chain G domain.

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.

Ile-Lys-Val-Ala-Val (IKVAV)-containing laminin alpha1 chain peptides form amyloid-like fibrils.

The Ile-Lys-Val-Ala-Val (IKVAV) sequence derived from laminin-1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV-containing peptide (LAM-L: AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108). The LAM-L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross-beta X-ray diffraction pattern. Further, infrared spectra of LAM-L suggested a beta-sheet structure. These results indicate that LAM-L forms amyloid-like fibrils. We also examined amyloid-like fibril formation of LAM-L analogs. The neurite outgrowth activity of the LAM-L analogs was closely related to their amyloid-like fibril formation.

Identification of cell binding sites in the laminin alpha5-chain G domain.

The laminins consist of at least 11 polypeptides (5 alpha-chains, 3 beta-chains, and 3 gamma-chains) specific to basement membranes. Here we investigate the biological activity associated with the G domain of the newly identified laminin alpha5-chain using 113 overlapping synthetic peptides (positions 2679-3635). Using HT-1080 cells, 21 peptides showed attachment activity either on peptide-coated tissue culture plates or to peptide-conjugated Sepharose beads. Heparin inhibited cell attachment to 16 peptides, while ethylenediaminetetraacetic acid exhibited no inhibitory activity. Peptides A5G-27, A5G-65, and A5G-71 showed the strongest cell attachment, with the minimum active core sequences of the peptides being GIIFFL, HQNMGSVNVSV, and YLQFVG, respectively. Furthermore, these 16 peptides were tested for their ability to stimulate neurite outgrowth in the PC12 cells. A5G-3, A5G-33, A5G-71, A5G-73, A5G-81, and A5G-101 were the only peptides of the 16 that demonstrated the ability to promote neurite outgrowth. These results demonstrate that synthetic peptides with alpha5-chain G domain primary amino acid sequences possess some of the same biological activities attributable to the whole laminin and the alpha5-chain G domain. Therefore, these peptides may be useful in the investigation of laminin-receptor interactions and possibly mechanisms of laminin signal transduction.