Gene expression of HPDLFs compared with HDFs by microarray analysis.

<p>Gene expression of HPDLFs compared with HDFs by microarray analysis.</p

RT-PCR and qRT-PCR of gene expression in MSCs exposed to CM from HPDLFs and CM from CXCL12 down-regulated HPDLFs.

<p>(A) RT-PCR of gene expression in MSCs. Expression of <i>CXCL12</i> was up-regulated in MSCs exposed to CM from HPDLFs at 24 h after incubation. <i>COL1A1</i> and <i>Alkaline phosphatase</i> were also up-regulated in MSCs. (B) qRT-PCR of gene expression in MSCs. <i>CXCL12</i> (a–c’) and <i>COL1A1</i> (b–c’) expressions were significantly up-regulated in MSCs exposed to CM from HPDLFs, as compared with gene expression of MSCs in control medium (a–b’ and b–b’). However, up-regulation was not observed in the expression of <i>COL3A1</i> (c–b’) and <i>PLAP1</i> (d–b’). MSCs exposed to CM from CXCL12-siRNA transfected HPDLFs (a–d’ and b–d’) or MSCs (a–b’ and b–b’) alone did not show up-regulation in expression of <i>CXCL12</i> and <i>COL1A1</i>. There were no significant differences in the expression of COL3A1 (c–b’–g’) and PLAP1 (d–b’–g’). <b>a’</b>: HPDLFs <b>b’</b>: MSCs (Medium only (control)) <b>c’</b>: MSCs (HPDLFs CM) <b>d’</b>: MSCs (CXCL12-siRNA transfected HPDLFs CM) <b>e’</b>: MSCs (HDFs CM) <b>f’</b>: MSCs (medium contained recombinant CXCL12) <b>g’</b>: MSCs (scramble siRNA-transfected HPDLFs CM). All samples compared to MSCs cultured in normal culture medium (b’). *denotes p values less than 0.01.</p

qRT-PCR and ELISA of expression of CXCL12 in HPDLFs, HDFs, HeLa and siRNA transfected HPDLFs.

<p>(A) qRT-PCR of CXCL12 in HPDLFs and HDFs. Expression of <i>CXCL12</i> in HPDLFs was significantly higher than in HDFs. <i>CXCL12</i>-siRNA transfected HPDLFs showed significantly lower expression of <i>CXCL12</i>. (B) ELISA of CXCL12 in HPDLFs and HDFs. CXCL12 protein secreted by HPDLFs, HDFs and HeLa cells in culture medium was measured by ELISA. Expression of CXCL12 in the supernatant from HPDLFs was significantly higher than that from HDFs and HeLa cells at the protein level. CXCL12-siRNA-transfected HPDLFs showed significantly lower expression, when compared with expression of CXCL12 in the HPDLFs. * and ** denote p values less than 0.05 and 0.01, respectively, by Bonferroni’s multiple comparison.</p

Results of RT-PCR and qRT-PCR.

<p>(A) RT-PCR of expression of <i>CXCL12</i> and connective tissue specific markers. <i>CXCL12</i> in HPDLFs showed distinctly higher expression when compared to HDFs and HeLa cells. (Bi, ii) qRT-PCR of expression of <i>CXCL12</i> in HPDLFs and HDFs from three donors for each population. There were no significant differences in the expression of <i>CXCL12</i> in the HPDLFs among three donors (HPDLFs1, HPDLFs2 and HPDLFs3) and also in the HDFs among three donors (HDFs1, HDFs2 and HDFs3) (P>0.05). (<b>C</b>): Effects of CXCL12-siRNA in HPDLFs from three donors and in HDFs from three donors. Significant down-regulation was commonly observed in HPDLFs among three donors (i). However, expression of CXCL12 was not influenced by CXCL12 siRNA in HDFs from three donors (ii).</p

Primer sequences used in this study.

<p>Primer sequences used in this study.</p

MSC migration assay.

<p>(Ai) Reactivity of MSCs from three donors to HPDLFs. Fluorescent microscopy showed no differences in the number of migrated cells of MSCs among the three donors. (Aii) Statistical analysis of migrated MSCs. There were no significant differences in the number of migrated cells of MSCs among the three donors. (B) Migration assay of MSCs. Fluorescent microscopy demonstrated that migrated MSCs by HPDLFs (<b>a</b>) were much more abundant than those by HDFs (<b>b</b>) and medium alone (c)<b>. a</b>: HPDLFs <b>b</b>: HDFs <b>c</b>: Medium only <b>d</b>: HPDLFs (MSCs were pre-treated with 5 µg/ml AMD3100) <b>e</b>: HPDLFs <b>(</b>MSCs were pre-treated with 100-µg/ml CXCR4-neutralizing antibody) <b>f</b>: HPDLFs (MSCs were pre-treated with 100-µg/ml IgG antibody) <b>g</b>: HPDLFs (transfected with CXCL12-siRNA) <b>h</b>: HPDLFs (transfected with scramble siRNA) <b>i</b>: recombinant CXCL12. <i>Bars, 100 µm</i>. Migrating MSCs by HPDLFs (<b>a</b>) were much more abundant than those by HDFs (<b>b</b>) and medium alone (c). (C) Statistical analysis of migrated MSCs. Number of migrating MSCs from HPDLFs (106.7±2.51 cells per field, P<0.01) was significantly higher than those from HDFs (39.3±2.30 cells per field, P<0.01), medium only (23.6±4.16 cells per field, P<0.01) and CXCL12-siRNA-transfected HPDLFs (25.3±0.57 cells per field, P<0.01). When MSCs were pre-treated with AMD3100 (15.6±1.52 cells per field, P<0.01) or CXCR4 antibody (25.6±9.07 cells per field, P<0.01), the number of migrated cells was significantly lower. *P<0.01 number of migrated MSCs by HPDLFs.</p

Potential to induce experimental infective endocarditis of oral LAB isolates.

<p>The potential to induce experimental infective endocarditis of 14 LAB (4 lactobacilli and 10 streptococci) isolates was evaluated in a rat model. The inducing potential was judged as positive when the bacteria administered were detected in either the blood or vegetation in more than 2 rats per group (6–7 rats). Rows in which there are less than 6 rats in a group show death due to either a technical mishap or infection during the experiment.</p><p>^aAveraged value of the number of detected bacteria.</p><p>^bPositive control.</p><p>^cBelow the detection limit.</p><p>Potential to induce experimental infective endocarditis of oral LAB isolates.</p

Identification of oral isolates based on 16S-rDNA partial sequencing.

<p>Identification of oral isolates based on 16S-rDNA partial sequencing.</p

Ability of oral isolates to produce VSCs and WIG.

<p>^aVSC (volatile sulfur compound) production <i>in vitro</i> was evaluated with an OralChroma. −, < detection limit; +/−, produced H₂S < 0.7 μg/10 mL/1 OD₅₅₀ or CH₃SH < 1.17 μg/10 mL/1 OD₅₅₀; +, produced H₂S > 0.7 μg/10 mL/1 OD₅₅₀ or CH₃SH > 1.17 μg/10 mL/1 OD₅₅₀; (CH₃)₂SH was below the detection limit in all strains.</p><p>^bWIG (water-insoluble glucan) production <i>in vitro</i>: −, non-producer; +/−, attachment of bacterial cells; +, producer.</p><p>Ability of oral isolates to produce VSCs and WIG.</p

List of probiotic candidates selected from oral LAB isolates.

<p>^a<i>Pg</i>, <i>P</i>. <i>gingivalis</i> ATCC 33277; <i>Pi</i>, <i>P</i>. <i>intermedia</i> ATCC 25611; <i>Sm</i>, <i>S</i>. <i>mutans</i> ATCC 25175; <i>Ss</i>, <i>S</i>. <i>sobrinus</i> ATCC 33478; <i>Aa</i>, <i>A</i>. <i>actinomycetemcomitans</i> Y4.</p><p>^b(−); Without neutralization, (+); with neutralization.</p><p>^cS-HA; salivary-coated hydroxylapatite.</p><p>^dCells originating from human buccal mucosa carcinoma.</p><p>^eCells originating from human tongue carcinoma.</p><p>List of probiotic candidates selected from oral LAB isolates.</p