Monitoring Bacterial Growth and LPS Production

Example 1

Growth of bacteria: A 1 ml aliquot of a 24 hour culture of E. coli ATCC 8739 was used to inoculate 100 ml of Luria-Bertani (LB) broth in a 250 ml baffled flask. This culture was then incubated at 37° C. with agitation (220 rpm) and sampled at 30 minute intervals. Samples were assessed for turbidity (OD₆₀₀) in a SpectraMax platereader M3 (Molecular Devices, Sunnydale, Calif.), which is one method of monitoring the growth and physiological state of microorganisms. The sample turbidity was then recorded and the samples were centrifuged at 5000 RPM for 10 min at room temperature. The supernatant, thereinafter referred to as “supernatant of bacterial culture”, was subsequently analyzed for LPS content using the procedure as described below.

Twenty ml aliquots of MTGE broth (Anaerobe Systems, Morgan Hill, Calif.) were inoculated with P. gingivalis ATCC 33277, P. pallens ATCC 700821, or P. nigrescens ATCC 25261. These cultures were incubated overnight in a Whitely A45 Anaerobic Workstation (Don Whitley Scientific, Frederick, Md.) at 37° C. with an 85:10:5 N₂:CO₂:H₂ gas ratio. One ml aliquots of these starter cultures were then used to inoculate 99 ml of membrane-Tryptone Glucose Extract (m-TGE) broth in a 250 ml baffled flask. These cultures were then incubated under agitation (200 rpm) as previously described and sampled at regular intervals. Samples were assessed for turbidity (OD₆₀₀) in a Tecan Infinite m200 Pro (Tecan Trading AG, Switzerland) and then centrifuged at 16,100×g for 10 min at room temperature. Supernatants were decanted and passed through a 0.22 μM filter prior to analysis for LPS content.

In the experiment, only OD600 was measured. For the sake of consistency in following experiments, we converted OD600 readings into bacterial numbers, even though the relationship between OD600 readings and bacterial numbers is varied for each bacterium. The number of bacteria was estimated based on spectrophotometer readings at OD₆₀₀ (OD₆₀₀ of 1.0=8×10⁸ cells/ml).

The Limulus Amebocyte Lysate Assay (LAL) is an assay to determine the total amount of lipopolysaccharide (LPS) in the sample tested (Pierce LAL Chromogenic Endotoxin Quantitation Kit, ThermoFischer Scientific, Waltham, Mass.). The assay was performed following manufacturer's instruction. Ninety-six-well microplates were first equilibrated in a heating block for 10 min at 37° C. Fifty μl each of standard or sample was dispensed into the microplate wells and incubated with plate covered for 5 min at 37° C. Then 50 μl LAL was added to each well. Plates were shaken gently and incubated for 10 min at 37° C. 100 μl of chromogenic substrate was added and incubated for 6 min at 37° C. Finally, 50 μl Stop Reagent was added and the absorbance was measured at 405-410 nm on Spectramax M3 platereader (Molecular Device, Sunnyvale, Calif.).

FIGS. 1A, 1C, and 1D show the ability of microbes to shed LPS as part of their normal growth cycle. This data shows the need to deliver chemistry to the subgingival plaque to effectively mitigate the LPS, since tooth brushing generally does not remove the subgingival plaque.

The LPS, as measured by the LAL kit reported in endotoxin unit per ml (EU/ml), was shed by the bacteria (E. coli K12) as depicted in FIG. 1A. The growth media began to be depleted of complex sugars around 120 minutes, as reflected in the bacterial growth curve in FIG. 1B, where the LPS shedding started to decline. This data gave a reason to believe that a mature biofilm/plaque could supply a constant level of LPS to the host cells, if food sources were present. The LPS would then have the ability to induce an inflammatory response from the host cells.

Importantly, LPS are secreted into the supernatant of bacterial culture (FIG. 1D). LPS also exists in bacterial walls (FIG. 1E). Again, this data further enforce the need to deliver chemistry to the subgingival plaque to effectively mitigate the LPS, since tooth brushing generally does not remove the subgingival plaque.

Example 2

Seven panelists, with at least three bleeding sites, took part in the testing. A licensed dental hygienist collected subgingival plaque samples. Samples were taken at the tooth/gum interface (buccal surfaces only) using care to avoid contact with the oral soft tissues. Six subgingival plaque sites were sampled from each panelist (3 healthy and 3 unhealthy sites). Unhealthy teeth had bleeding sites with pockets greater than 3 mm and healthy sites had no bleeding with pocket depth less than 2 mm Prior to sampling, panelists were instructed to abstain for 12 hours from oral hygiene and refrain from eating, chewing gum, drinking (except small sips of water). Next, panelists had their marginal plaque collected with a curette at the sampling sites. Then, from the same site, subgingival plaque samples were collected with 3 consecutive paper points as shown in FIG. 1F. The sampling sites were isolated with cotton rolls and gently air-dried. Paper points (PROFLOW incorporated, Amityville, N.Y.) were gently placed for 10 seconds into the pocket until a minimum of resistance was felt. After 10 seconds, paper points were removed and placed into pre-labeled 1.5 ml tubes. The same sampling procedure was repeated with 2 more paper points (paper points go into separate tubes). The first, second and third sample paper points from a healthy site of all panelists were pooled separately into three tubes, labeled as paper point 1, 2 and 3, respectively. Similarly the unhealthy site samples were also pooled.

TABLE 1 showed that unhealthy dental plaques contained more endotoxins than the healthy dental plaques. One m1 PBS was added to each pooled sample in the 1.5 ml tube. Bacteria were lysed in a MolBio Fast Prep bead beater (MP Biomedicals, Santa Ana, Calif.). Samples were centrifuged for 10 min at 10,000 RPM at 4° C., supernatants were collected and analyzed with LAL assay kits following manufacturer's instruction as described in EXAMPLE 1.

It was expected that the marginal plaques in unhealthy sites had more endotoxins than those in the healthy sites (TABLE1) within the same subjects. Three samples were taken from subgingival pockets with three paper points sequentially, named paper point 1, 2 and 3. Again, the subgingival plaques taken by the paper point 1 had more endotoxins in the unhealthy sites than in the healthy sites (TABLE 1). The same is true for the samples taken by paper point 2 and 3. Importantly, dental plaques in the unhealthy subgingival pockets possessed more endotoxins than plaques from healthy pockets. This may explain why unhealthy gingiva are prone to bleeding upon probing.

Example 3

The LAL assay, as described in EXAMPLE 1, was modified for development of technology which inhibits LPS from activating a proenzyme in the LAL assay. The Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit is a quantitative endpoint assay for the detection of LPS, which catalyzes the activation of a proenzyme in the modified Limulus Amebocyte Lysate (LAL). The activated proenzyme then splits p-Nitroaniline (pNA) from the colorless substrate, Ac-Ile-Glu-Ala-Arg-pNA. The product pNA is photometrically measured at 405-410 nm. If SnF2 binds to LPS, the latter can't react with the proenzyme in the LAL kit. Consequently, the proenzyme is not activated, and the colorless substrate Ac-Ile-Glu-Ala-Arg-pNA will not split and no color product is produced. P. gingivalis LPS 1690 (1 ng/ml), or E. coli LPS (1 ng/ml), and stannous fluoride and other materials (50 and 500 μM), as listed in TABLE 2, were dissolved in endotoxin-free water. Then 50 μl LAL was added to each well. Plates were shaken gently and incubated for 10 min at 37° C. 100 μl of chromogenic substrate was added and incubated for 6 min at 37° C. Finally, 50 μl Stop Reagent was added and the absorbance was measured at 405-410 nm on Spectramax M3 plate reader (Molecular Device, Sunnyvale, Calif.).

As shown in TABLE 2, SnF2 and some other compounds inhibited LPS activities in LAL assays

Example 5

Reporter gene cell lines, human HEK 293T cells, were purchased from Invivogen of San Diego, Calif. The HEK 293T cells were stably transfected with at least two exogenous genes, a TLR4 structural gene, and a SEAP reporter gene, which is under the control of NFkB transcriptional factors. The cell line is named here as TLR4-SEAP. The reporter gene encodes a secreted enzyme, called embryonic alkaline phosphatase or SEAP. The SEAP reporter is placed under the control of the interferon-β minimal promoter fused to five NFkB and AP-1-binding sites. Furthermore, the TLR4-SEAP cell line also contains a CD14 co-receptor gene, which is needed to transfer LPS to TLR4 receptors. The recombinant TLR binds its ligand, or distinct pathogen-associated molecule, initiates a chain of responses, leading to recruitment of NFkB and AP1 transcription factors to the reporter gene promoter, which induce expression of SEAP.

Cell culture and treatment: 500,000 gene reporter cells were grown and maintained in 15 ml growth medium, comprised of DMEM medium supplemented with 10% fetal calf serum in T75 flasks for three days at 37° C., 5% CO₂, and 95% humidity. For treatment, wells of a 96-well plate were seeded with 10,000 cells/well in 100 μL of growth medium. The cells were incubated for 72 hours at 37° C., 5% CO₂, and 95% humidity until day 4. On day 4, medium was changed to assay medium (90 μl), which is the DMEM medium without fetal calf serum. LPS, bacteria and the culture medium of bacterial growth, as described in EXAMPLE 1, were first resolved or mixed with the assay medium. 10 μl of the bacteria, LPS and culture medium of bacterial growth were added to the TLR4-SEAP cells. Samples were taken 24 hours later, following addition of LPS, bacteria, and culture medium. Expression of the reporter gene (SEAP) was quantified with a commercially available kit (SEAP Reporter Gene Assay of Cayman Chemical Co., Ann Arbor, Mich.).

EC50 was calculated using GraphPad Prism software (GraphPad Software, La Jolla, Calif.). Samples with lower EC50 are more potent in activating the TLR4 reporter gene than those with higher EC50. As shown in FIG. 2A, LPS from E. coli has lower EC50 than P. gingivalis, thus, was far more potent than P. gingivalis (Pg). Salmonella Minnesota LPS is not as potent as that of E. coli, but is far more potent than those of P. gingivalis LPS 1690 and 1435. Each species of bacteria produces multiple forms of LPS. Each form of LPS from the same species of bacteria has different potency in stimulating TLR4-downstream signaling pathways. For example, Pg 1690 LPS is more potent than Pg1435/50. LPS is a component in bacterial cell walls. Likely, E. coli cell wall is more virulent in inducing production of proinflammatory cytokines in host cells than P. gingivalis when they are in direct contact with host blood cells. P. gingivalis had far higher EC50 than P. pallens and P. nigrescens as shown in FIG. 2B in stimulating TLR4 reporter gene expression, suggesting that P. pallens and P. nigrescens are more likely to promote production of proinflammatory cytokines in host cells than P. gingivalis.

Bacteria release LPS into the supernatant of bacterial culture. As shown in FIG. 2C, the supernatant of P. pallens has an EC50 that is similar to that of P. nigrescens, but far lower than that of P. gingivalis, in stimulating expression of TLR4 reporter gene. Again, those results imply that the products of P. pallens and P. nigrescens are more likely to promote production of proinflammatory cytokines in host cells than those of P. gingivalis.

Example 6

Stannous fluoride is a leading anti-gingivitis technology in P&G toothpaste products. Tests were conducted to understand whether stannous fluoride could reduce LPS's ability to trigger proinflammatory responses in host cells. TLR4-SEAP reporter cells were prepared using the same conditions as described in EXAMPLE 5 in the presence or absence of LPS. Production of SEAP was quantified also as described in EXAMPLE 5.

FIG. 3 shows the effect of stannous at various concentrations from 62.5 uM to 1,000 uM on 100 ng/ml E. coli LPS, as reported by activation of TLR-4. At stannous concentrations of 500 uM or higher, the level of E. coli induction of TLR-4 was decreased.

FIG. 4 shows the effects of stannous at various concentrations from 62.5 uM to 1,000 uM on P. gingivalis LPS, as reported by activation of TLR-2. At stannous concentrations of 1000 uM, the level of P. gingivalis induction of TLR-2 was decreased.

The data in FIG. 5 shows reduction of LPS activity by the stannous ion, from a stannous fluoride salt. The data showed that stannous fluoride, at 1.6 mM and 3.2 mM, reduce about 50% of P. gingivalis LPS (500 ng/ml) activation on the TLR4 reporter system (One asterisk means P<0.05, two asterisks mean P<0.01).

Example 7

The method described in EXAMPLE 5 is effective at determining the potency of LPS from different bacteria. The same method was used to determine the EC50 of clinical samples, as described in EXAMPLE 2. As shown in FIG. 6, dental plaques from unhealthy sites had a smaller EC50 than those from healthy sites, suggesting the dental plaques from unhealthy sites contain more virulence factors.

The same method described in EXAMPLE 5 was used to examine the clinical samples in another study. A clinical study was conducted to evaluate sample collection methods and measurement procedures. It was a controlled, examiner-blind study. Forty panelists met the inclusion criteria, wherein in order to be included in the study, each panelist must:

• • Provide written informed consent to participate in the study;
  • Be 18 years of age or older;
  • Agree not to participate in any other oral/dental product studies during the course of this study;
  • Agree to delay any elective dentistry (including dental prophylaxis) until the study has been completed;
  • Agree to refrain from any form of non-specified oral hygiene during the treatment periods, including but not limited to the use of products such as floss or whitening products;
  • Agree to return for all scheduled visits and follow study procedures;
  • Must have at least 16 natural teeth;
  • Be in good general health, as determined by the Investigator/designee based on a review of the health history/update for participation in the study.

For Unhealthy Group (high bleeder group):

• • Have at least 20 bleeding sites (sites with a score of 1 or 2 on the GBI index); Have minimum 3 sampling sites with bleeding and pocket depth >3 mm but not deeper than 4 mm;
  • Have minimum 3 sampling sites without bleeding and with pocket depth <2 mm

For Healthy Group (low bleeder group):

• • Have maximum 3 bleeding sites (sites with a score of 1 or 2 on the GBI index);
  • No pockets deeper than 2 mm. Twenty (20) panelists were qualified as healthy—with up to 3 bleeding sites and with all pockets less than or equal to 2 mm deep and twenty (20) panelists were qualified as unhealthy—with greater than 20 bleeding sites with at least 3 pockets greater than or equal to 3 mm but not deeper than 4 mm with bleeding, and at least 3 pockets less than or equal to 2 mm deep with no bleeding for sampling. All panelists had up to 6 sites identified as “sampling sites.” The “sampling sites” had supragingival and subgingival plaque collected at Baseline, Week 2 and Week 4. Subgingival plaque samples were taken from a gingival sulcus from the pre-identified sites. Prior to sample collection, the site had supragingival plaque removed with a curette. The site was dried and subgingival plaque samples were collected with another dental curette (e.g., Gracey 13/14, 15/16, 11/12, 7/8, ½.) Each Gracey curette is designed to adapt to a specific area or tooth surface. For example, Gracey 13/14 is designed to adapt to the distal surfaces of posterior teeth. Samples from each site were placed in a pre-labeled 2.0 ml sterile tube containing 300 μl of DPBS buffer with about 50 of sterile 1 mm glass beads. Samples were stored at 4° C. The subgingival samples were stored at −80° C. until analyzed. The samples were thawed at room temperature and dispersed in a TissueLyser II (Qiagen, Valencia, Calif., USA) at 30 shakes per second for 3 min. Protein concentrations of the dispersed subgingival samples were measured using a Pierce microBCA Protein kit (ThermoFisher Scientific, Grand Island, N.Y., USA) following the manufacturer's instruction.

Oral lavage samples were collected at wake up (one per panelist) by rinsing with 4 ml of water for 30 seconds and then expectorating the contents of the mouth into a centrifuge tube. These samples were frozen at home until they were brought into the site in a cold pack. Each panelist collected up to 15 samples throughout the study. Saliva samples were frozen at −70° C. from submission.

All panelists were given investigational products: Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush. Panelists continued their regular oral hygiene routine, and did not use any new products starting from the baseline to the end of four week treatment study. During the four week treatment period, panelists brushed their teeth twice daily, morning and evening, in their customary manner using the assigned dentifrice and soft manual toothbrush.

The subgingival plaques from the above clinical study were applied to the TLR4 reporter cells in a procedure as described in EXAMPLE 5. FIG. 7A shows the results of a four-week study of 40 panelists going from baseline out over four weeks of treatment with Crest ProHealth Clinical toothpaste. The subgingival plaque samples in bleeding sites on the high bleeders group stimulated high expression of TLR4 reporter gene. More virulence in a sample elicits higher RLU (relative luminescent units) readings in the TLR4 reporter gene assay. As shown in FIG. 7A, the baseline samples of the high bleeders group had higher RLU than those of the low bleeders on both the bleeding and non-bleeding sites. After treatment with Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks, the virulence was reduced at week 4 in both high and lower bleeders groups at both bleeding and non-bleeding sites.

The oral lavage samples were applied to the TLR4 reporter cells in a procedure as described in EXAMPLE 5. As shown in FIG. 7B, oral lavage (Healthy vs. Gingivitis) samples were evaluated in the TLR4-SEAP reporter assay. The baseline samples of the high bleeders group had higher RLU than those of the low bleeders. After treatment with Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks, the virulence was reduced at week 4 in the high bleeder group.

Example 8

The reporter gene cell lines, human HEK 293T cells, were purchased from Invivogen of San Diego, Calif. The HEK 293T cells were stably transfected with at least two exogenous genes, a TLR2 structural gene, and SEAP reporter gene which is under the control of NFkB transcriptional factors. The cell line is named here as TLR2-SEAP. The reporter gene encodes a secreted enzyme, called embryonic alkaline phosphatase or SEAP. The SEAP reporter is placed under the control of the interferon-β minimal promoter fused to five NFkB and AP-1-binding sites. Furthermore, a CD14 co-receptor gene was transfected into the reporter gene cells expressing TLR2, as CD14 has been identified as a co-receptor for TLR2 ligands to enhance the TLR response. The CD14 co-receptor is needed to transfer LTA to TLR2 receptors. The recombinant TLR2 binds its ligand, or distinct pathogen-associated molecule, initiates a chain of responses, leading to recruitment of NFkB and AP1 transcription factors to the reporter gene promoter, which induce expression of SEAP.

Cell culture and treatment: 500,000 gene reporter cells were grown and maintained in 15 ml growth medium, comprising DMEM medium supplemented with 10% fetal calf serum in T75 flasks for three days at 37° C., 5% CO₂, and 95% humidity. For treatment with LTA, wells of a 96-well plate were seeded with 10,000 cells/well in 100 μL of growth medium. The cells were incubated for 72 hours at 37° C., 5% CO₂, and 95% humidity until day 4. On day 4, medium (100 μL) was changed to DMEM medium without fetal calf serum. LTA, LPS and bacterial cells, as described in EXAMPLE 7, were added. Samples were taken 24 hours later, following addition of samples. Expression of the reporter gene (SEAP) was quantified with a commercially available kit (SEAP Reporter Gene Assay of Cayman Chemical Co., Ann Arbor, Mich.).

As shown in FIGS. 8A, 8B, 8C and 8D, LTA, LPS, bacteria and the supernatant of bacterial culture could bind to TLR2 and activate TLR2 downstream signaling pathways in a dose-dependent manner. As shown in FIG. 8A, B. subtilis (BS) LTA is more potent than that of Enterococccus hirae. As shown in FIG. 8B, P. gingivalis LPS also activated expression of the TLR2 reporter gene. For example, Pg1690, as shown in FIG. 8B, activated TLR2-SEAP signal transduction, and stimulated SEAP production. But as shown in FIG. 8B, E. coli LPS did not activate the TLR2-SEAP reporter cells. It should also be noted that P. pallens, P. nigrescens and P. gingivalis have similar EC50 in stimulating expression of TLR2 reporter gene (FIG. 8C). However, the released TLR2 ligands from the three different bacteria have very different EC50 on activation of TLR2 reporter gene (FIG. 8D).

Example 9

The method described in EXAMPLE 8 is effective in determining the EC50 of LTA and other TLR2 ligands from different bacteria. The same method was used to determine the EC50 of clinical samples, as described in EXAMPLE 2. As shown in FIG. 9, dental plaques from unhealthy (bleeding) sites had smaller EC50 than those from healthy (non-bleeding) sites, suggesting the dental plaques from unhealthy sites contain more virulence factors.

Clinical samples as described for FIG. 7A of EXAMPLE 7 were examined using the TLR2-SEAP reporter gene assay. The results are shown in FIG. 10A. The subgingival samples in unhealthy (bleeding) sites from the unhealthy group (high bleeders) had more virulence factors than other sites. The baseline samples of the high bleeders group had higher RLU than those of the low bleeders on both the bleeding and non-bleeding sites. After treatment with Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks, the virulence was reduced at week 4 in both high and low bleeders groups at both bleeding sites.

The clinical samples as described for FIG. 7B of EXAMPLE 7 were examined using the TLR2-SEAP reporter gene assay. As shown in FIG. 10B, oral lavage (Healthy vs. Gingivitis) was evaluated. After treatment with Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks, the virulence was reduced at week 4 in the high bleeder group.

Example 11

Bacterial cell wall and membrane components are recognized by TLR2. TLR2 recognizes the microbial motifs PGN (peptidoglycan)/lipoproteins/dectin and LPS. TLR1 and TLR6 form heterodimers with TLR2 and bind to triacylated lipoproteins and diacylated lipoproteins, respectively. THP1 NFkB-SEAP and IRF-Lucia™ Reporter Monocytes were purchased from Invivogen, San Diego, Calif. THP1-Dual cells were derived from the human THP-1 monocyte cell line by stable integration of two inducible reporter constructs. THP1-Dual cells feature the Lucia gene under the control of an ISG54 (interferon-stimulated gene) minimal promoter in conjunction with five interferon-stimulated response elements. THP1-Dual cells also express a SEAP reporter gene driven by an IFN-b minimal promoter fused to five copies of the NF-kB consensus transcriptional response element and three copies of the c-Rel binding site. As a result, THP1-Dual cells allow the simultaneous study of the NFkB pathway, by monitoring the activity of SEAP, and the interferon regulatory factor (IRF) pathway, by assessing the activity of Lucia (IRF-Luc). Both reporter proteins are readily measurable in the cell culture supernatant. This THP-1 cell line possesses functional TLR1, TLR2, TLR4, TLR5, TLR6 and TLR8, purchased from Invivogen. TLR4 senses LPS from Gram-negative bacteria while TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, TLR8 detects long single-stranded RNA.

Culture and treatment: The THP1-dual cells were cultured in 15 ml growth medium (RPMI 1640 with 10% heat-inactivated fetal bovine serum) in a T75 flask at 37° C. and 5% CO₂. Cells were passed every 3 to 4 days by inoculating 300,000-500,000 cells/ml into a fresh T75 flask with 15 ml of fresh growth medium. To determine the effect of bacterial components on reporter gene expression, wells in 96-well plates were seeded at 100,000 cells in 90 μl of growth medium. 10 μl of bacterial wall and membrane components, or heat-killed whole bacteria, were added to each well. After incubation for 18 hours at 37° C. and 5% CO₂, secreted luciferase and SEAP were quantified with commercially available assay kits (QUANTI-Luc of Invivogen, San Diego, Calif. for luciferase; SEAP Reporter Gene Assay of Cayman Chemical Co., Ann Arbor, Mich. for SEAP).

DHP1-dual reporter cells were treated with three different preparations of LPS as shown in FIG. 12A. All three LPS (ng/ml) activated production of NFkB-SEAP reporter genes in a dose-dependent manner. In addition, Pg 1690 LPS and E. coli LPS also stimulated expression of the IRF-luciferase reporter gene. TLR4 ligands, upon binding to TLR4 receptors, activate at least two signaling pathways. One is a common pathway NFkB-SEAP, which can be activated by all TLR ligands upon binding to their specific receptors. For example, TLR2 ligand, LTA, can bind to TLR2 receptors and activate the NFkB-SEAP signaling pathway. Similarly, TLR4 ligand, LPS, upon binding to TLR4 receptors, also is able to activate the NFkB-SEAP signaling transduction. As shown in FIG. 12A, E. coli LPS is a more potent ligand than P. gingivalis 1690 LPS on activation of both NFkB-SEAP and IRF-luciferase signaling transduction. THP-1 cells produce several functional TLR receptors. And all TLR receptors can activate the NFkB pathway, thus promoting expression of the NFkB-SEAP reporter gene. The reading of NFkB-SEAP is the collective actions of all TLR receptors, such as TLR2, TLR1, TLR6 and TLR4. All LPS from different bacteria stimulated NFkB-SEAP reporter gene. IRF-luciferase reporter gene, on the other hand, is driven by a limited number of TLR receptors, primarily TLR3, TLR4, TLR7, TLR8 and TLR9. Both P. gingivalis LPS 1690 and E. coli LPS stimulated expression of IRF-luciferase in a dose-dependent fashion.

The THP-1 reporter cells were used to examine the clinical samples as described for FIG. 7B of EXAMPLE 7. As shown in FIG. 12B, oral lavage (Healthy vs. Gingivitis) was evaluated using the IRF-Luc reporter gene in THP-1 cells. After treatment with Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks, the virulence was reduced at week 4 in both high and lower bleeders groups.

The THP-1 reporter cells were used to examine the clinical samples as described for FIG. 7A of EXAMPLE 7. As shown in FIG. 12C, the subgingival Plaque (Healthy vs. Gingivitis) was examined using the NFkB reporter gene in THP-1 cells. The baseline samples of the high bleeders group had higher RLU than those of the low bleeders. After treatment with Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks, the virulence was reduced at week 4 in the bleeding sites in both high and lower bleeders groups.

Example 12

THP1 dual reporter cells also express TLR2, TLR1 and TLR6 receptors. Bacterial cell wall and some membrane components are recognized by TLR2, TLR1 and TLR6. TLR2 recognizes the microbial motifs PGN (peptidoglycan)/lipoproteins/dectin and LPS. To determine whether LTA from different bacteria have different effects on stimulating NFkB-SEAP reporter gene expression in the THP1 dual reporter cells, the cells were prepared and treated in the same procedures as described in EXAMPLE 11. As shown in FIG. 13, LTA from both B. subtilis and S. aureus had similar potency in promoting reporter gene expression in the THP1 dual reporter cells.

Example 14

A randomized, two-group clinical study was conducted with 69 panelists (35 in the negative control group and 34 in the test regimen group). Panelists were 39 years old on average, ranging from 20 to 69, and 46% of the panelists were female. Treatment groups were well balanced, since there were no statistically significant (p≥0.395) differences for demographic characteristics (age, ethnicity, gender) or starting measurements for Gingival Bleeding Index (GBI); mean=29.957 with at least 20 bleeding sites, and Modified Gingival Index (MGI); mean=2.086. All sixty-nine panelists attended each visit and completed the treatment process. The following treatment groups were compared over a 6-week period:

Test regimen: Crest® Pro-Health Clinical Plaque Control (0.454% stannous fluoride) dentifrice, Oral-B® Professional Care 1000 with Precision Clean brush head and Crest® Pro-Health Refreshing Clean Mint (0.07% CPC) mouth rinse. Control regimen: Crest® Cavity Protection (0.243% sodium fluoride) dentifrice and Oral-B® Indicator Soft Manual toothbrush.

Dental plaques were collected from the same panelists in the test regimen in the clinical study as described in EXAMPLE 2. A supragingival sample was taken from each panelist with a sterile curette at the tooth/gum interface, using care to avoid contact with the oral soft tissue. Plaques were sampled from all available natural teeth (upper arch only) until no plaque was visible. Following sampling, the plaque samples were placed into a pre-labeled (panelist ID, sample initials, visit, and date) Eppendorf tube with 1 ml of PBS/Glycerol buffer and about 50 of sterile 1 mm glass beads, stored on ice until all samples were collected. The samples were then transferred to a −70° C. freezer for storage until further processing. Genomic DNA was isolated from supragingival plaque samples using QIAamp® genomic DNA kits (Qiagen, Germany) following manufacturer's instruction. Metasequencing was carried out at BGI Americas Corporation (Cambridge, Mass.). All data were analyzed at Global Biotech of Procter & Gamble Company in Mason, Ohio.

Clinical measurements: Bleeding sites (GBI) were decreased in the test regimen significantly on week 1, 3 and 6 in comparison to the control regimen (FIG. 14). Similarly, Inflammation (MGI) grades also decreased in the test regimen (FIG. 14).

Genomic DNA of the supragingival plaques in the test regimen was sequenced. As shown in FIG. 15, abundance of certain bacteria in the supragingival plaques changed in the six week treatments. Certain bacteria, such as Porphyromonas sp oral taxon 279 and Prevotella pallens, were decreased in weeks 1 and 3 (FIG. 15). The amount of each bacterial species was plotted over the four time periods of the treatment. The amount of certain bacteria, such as Peptostreptococcus stomatis and Prevotella intermedia, was reduced during the six week of treatment as shown in FIG. 15. This is also shown in FIG. 16A-1, FIG. 16A-2, and FIG. 16A-3.

Example 15

In the same clinical study as described in EXAMPLE 14, gingival-brush samples were collected from the same panelists as in EXAMPLE 14. Before sampling, panelists rinsed their mouths for 30 seconds with water. A dental hygienist then sampled the area just above the gumline using a buccal swab brush (Epicentre Biotechnologies cat.# MB100SP). The swab was immediately placed into 1 ml extraction buffer [PBS, 0.25M NaCl, 1× Halt™ Protease Inhibitor Single-Use Cocktail (Lifetechnologies, Grand Island, N.Y.)] in a 1.5 ml Eppendorf tube vortexed for 30 seconds, and immediately frozen on dry ice and stored in a −80 C freezer until analysis. The samples were taken out of freezer, thawed and extracted by placing the samples on a tube shaker for 30 minutes at 4° C. The tubes were centrifuged at 15000 RPM for 10 min in Eppendorf Centrifuge 5417R (Eppendorf, Ontario, Canada) to pellet any debris. The extract (800 μl) was analyzed for protein concentrations using the Bio-Rad protein assay (BioRad, Hercules, Calif.).

Forty proteins were measured in the gingival samples using V-PLEX Human Biomarker 40-Plex Kit (Meso Scale Diagnostics Rockville, Md.). The assay was performed following the manufacturer's instruction.

Among the proteins measured in the gingival samples, most proteins in the Proinflammatory Panel 1 (human), Cytokine Panel 1 (human), Chemokine Panel 1 (human), Angiogenesis Panel 1 (human), and Vascular Injury Panel 2 (human) had significant changes in their abundance during the 6-week treatment (TABLE 6). Those include FN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α, GM-CSF, IL-5, IL-16, IL-7, IL-12/IL-23p40, IL-1α, VEGF-A, IL-17A, IL-15, TNF-β, IL-8 (HA), MCP-1, MCP-4, Eotaxin, IP-10, MDC, Eotaxin-3, TARC, MIP-1α, MIP-1β, VEGF-C, VEGF-D, Tie-2, Flt-1/VEGFR1, PlGF, FGF (basic), SAA, CRP, VCAM-1, and ICAM-1.

Example 16

The same gingival-brush samples as described in EXAMPLE 15 were used for metabonomic analyses. Fourteen panelists were selected randomly from each treatment group to determine if any metabolite concentrations were changed in gingival samples during the first 3 weeks of treatment. Both baseline and week 3 samples were sent to Metabolon, Inc. (Durham, N.C.) for metabonomic measurement. 170 metabolites were identified and quantified. As shown in TABLE 7, some metabolite concentrations were changed during the first 3 weeks of treatment. Citrulline concentrations in the gingival samples were reduced after three weeks of treatment in the treatment regimen group. Similarly, ornithine was also reduced in the treatment regimen group after three weeks of treatment. Reduction of citrulline and ornithine was likely associated with alleviation of gingivitis.

Example 17

Quantitation of citrulline and ornithine from the extracts of the Gingival-brush samples was conducted using gradient hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS). Gingival-brush samples were obtained from the same human panelists in the clinical study as described in EXAMPLE 14, and were placed into extraction buffer as described in EXAMPLE 15. The supernatants were subject to both HILIC/MS/MS and BCA analysis. For free citrulline and ornithine analysis, the extracts of the Gingival-brush samples were analyzed either directly (50 μl undiluted sample solution) in 50/50 acetonitrile/ultra-pure water with 0.754% formic acid or diluted fivefold. For total citrulline and ornithine analysis, the extracts of the Gingival-brush samples were first hydrolyzed using 6 N HCl (50 μL of extract with 450 μL of 6N HCl), no shaking, and placed on a hot plate at 110° C. for 16 hours. The hydrolyzed samples were then dried down under vacuum at room temperature (Savant speedvac of Lifetechnology, Grand Island, N.Y.) and then reconstituted in 1 ml of dilution solution (50/50 acetonitrile/ultra-pure water with 0.754% formic acid) for analysis. The standards and the samples were analyzed using gradient hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS). Analytes and the corresponding ISTDs (stable isotope labeled internal standard) were monitored by electrospray ionization (ESI) in positive mode using the selected-reaction-monitoring schemes shown in TABLE 8. A standard curve was constructed by plotting the signal, defined here as the peak area ratio (peak area analyte/peak area ISTD), for each standard versus the mass of each analyte for the corresponding standard. The mass of each analyte in the calibration standards and Gingival-brush extract samples were then back-calculated using the generated regression equation. The concentration of protein bound citrulline or ornithine was calculated as the result of subtracting the concentration of free citrulline or ornithine from the concentration of total citrulline or ornithine, respectively. The result was reported as the concentration of citrulline or ornithine or the result was standardized by dividing by the amount of citrulline or ornithine by the amount of the total proteins that were found in the extract.

All samples from all panelists of the Test regimen [Crest® Pro-Health Clinical Plaque Control (0.454% stannous fluoride) dentifrice, Oral-B® Professional Care 1000 with Precision Clean brush head and Crest® Pro-Health Refreshing Clean Mint (0.07% CPC) mouth rinse] were analyzed. As shown in FIG. 16, citrulline levels reduced rapidly in the first week of treatment, and then continued to decline gradually in weeks 3 and 6 of treatment. These results are consistent with clinical observations, where gingival bleeding sites (GBI) and the gingival inflammation (MGI) were reduced over the 6-week treatment period.

Example 18

The same samples as described in EXAMPLE 17 were analyzed using procedures as described in EXAMPLE 17. Gingivitis was treated for 6 weeks. Baseline (BL) represents diseased status. Symptoms of gingivitis were alleviated from week 1 to week 6 treatments. Protein bound ornithine (the difference between total and the free ornithine) was higher in gingivitis as shown in FIG. 17. Protein bound ornithine was reduced gradually as gingivitis was decreased in severity.

Example 19

Gingival samples were collected as described in EXAMPLES 15, from the same panelists as in EXAMPLE 15, and were used to examine the expression of genes during 6 weeks of treatments with Test regimen [Crest® Pro-Health Clinical Plaque Control (0.454% stannous fluoride) dentifrice, Oral-B® Professional Care 1000 with Precision Clean brush head and Crest® Pro-Health Refreshing Clean Mint (0.07% CPC) mouth rinse] and Control regimen [Crest® Cavity Protection (0.243% sodium fluoride) dentifrice and Oral-B® Indicator Soft Manual toothbrush].

After harvesting the samples, the brush was completely immersed in the RNAlater solution (1 ml in a 1.5 ml Eppendorf tube) for keeping RNA from degrading during transport and storage (Qiagen, Valencia, Calif.). The microcentrifuge tubes were vortexed/mixed for 30 seconds, immediately frozen on dry ice, stored and transferred on dry ice to the lab for biomarker analysis. RNA isolation and microarray analysis were performed as described previously in a publication (Offenbacher S, Barros S P, Paquette D W, Winston J L, Biesbrock A R, Thomason R G, Gibb R D, Fulmer A W, Tiesman J P, Juhlin K D, Wang S L, Reichling T D, Chen K S, Ho B. J Periodontol. 2009 December; 80(12):1963-82. doi: 10.1902/jop.2009.080645. Gingival transcriptome patterns during induction and resolution of experimental gingivitis in humans).

The ornithine-citrulline-arginine cycle consists of four enzymes (FIG. 18). The main feature of the cycle is that three amino acids (arginine, ornithine, and citrulline) can be converted to each other. The first enzyme is ornithine transcarbamoylase, which transfers a carbamoyl group from carbamoyl phosphate to ornithine to generate citrulline. This reaction occurs in the matrix of the mitochondria. Expression of ornithine transcarbamoylase was reduced in the treatment (FIG. 19). The second enzyme is argininosuccinate synthetase. This enzyme uses ATP to activate citrulline by forming a citrullyl-AMP intermediate, which is attacked by the amino group of an aspartate residue to generate argininosuccinate. This and subsequent two reactions occur in the cytosol. Again, expression of argininosuccinate synthetase decreased during the treatment. The third enzyme is argininosuccinate lyase, which catalyzes cleavage of argininosuccinate into fumarate and arginine. The last enzyme is argininase. Argininases cleave arginine to produce urea and ornithine. In a contrast to the decreased expression of ornithine transcarbamoylase and argininosuccinate synthetase genes, argininase I and II increased (FIG. 19).

Arginine is also a substrate for nitric oxide synthase, which oxidizes arginine to produce citrulline and nitric oxide. Expression of nitric oxide synthase gene increased too (FIG. 19).

Example 20

Experimental gingivitis: Another clinical study was carried out to determine whether citrulline is increased in experimentally induced gingivitis in healthy human panelists. This was a case-control study enrolling 60 panelists. The study population included two groups as follows: Group 1 or high bleeders group, thirty (30) panelists with at least 20 bleeding sites, where bleeding is a GBI site score of 1 or 2 at baseline. Group 2 or low bleeders group, thirty (30) panelists with 2 or less bleeding sites, where bleeding is a GBI site score of 1 or 2.

The study consisted of two Phases: Health/Rigorous Hygiene Phase with dental prophylaxis, polishing and rigorous oral hygiene; and Induced Gingivitis Phase without oral hygiene. At the Screening visit, panelists underwent an oral soft tissue assessment and had a gingivitis evaluation (Modified Gingival Index (MGI) and Gingival Bleeding Index (GBI). At Visit 2 qualifying panelists received an oral soft tissue exam followed by a gingivitis evaluation and gingival plaques and gum swabs were collected for the qPCR, protein and RNA host biomarker analysis. Following that, all panelists received dental prophylaxis and entered the Health/Rigorous Hygiene Phase, lasting two weeks. After two weeks of rigorous hygiene, panelists entered the Induced Gingivitis Phase, lasting for three weeks. Oral soft tissue exams and gingivitis were re-evaluated and all samples (gum swabs) were collected at Baseline, WK0 and WK2.

Gingival sample collection—A gum swab was collected from each side of the upper arch using the procedures as described in EXAMPLE 15. Gum swabs were collected close to the gum line from the buccal sites only (preferably from four adjacent teeth—preferably from premolar and molar areas). Panelists rinsed for 30 seconds with 15 ml of Listerine rinse to clean the surface of sampling area. After the Listerine rinse, panelists rinsed for 30 seconds with 20 ml of water. Following that, selected sites were isolated with a cotton roll and gently dried with an air syringe and two gum swabs were taken with collection brushes/swabs from the gingiva region close to the gumline of the selected teeth. The samples were placed in a pre-labeled (panelist ID, sample ID, visit, and date) 1.5 ml micro-centrifuge tube containing 800 ul DPBS (Dulbecco's phosphate-buffered saline) (Lifetechnologies, Grand Island, N.Y.) with protease inhibitors, including AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) 2 mM, aprotinin 0.3 μM, Bestatin 130 μM, EDTA (Ethylenediaminetetraacetic acid) 1 mM, E-64 1 μM, and leupeptin 1 μM. The vials were vortexed/mixed for 30 seconds, immediately frozen on dry ice, stored and transferred on dry ice to the lab for biomarker analysis. Samples from three visits were analyzed using the procedures described in EXAMPLE 17, and shown in FIG. 20. Those three visits were baseline, Week 0, (right after the Health/Rigorous Hygiene Phase and before the induced gingivitis phase) and week 2 (at the end of Induced Gingivitis Phase). Free citrulline levels were low in both the high and low bleeders groups at the baseline and week 0, but rose quickly in the induced gingivitis in both groups at week 2.

Example 21

The same procedures were used as described in EXAMPLE 17. The samples were the same as described in EXAMPLE 20. Protein bound citrulline was lower at the baseline than that at week 0 in both high and low bleeders groups as shown in FIG. 21 in gingival tissue. It was low in experimental gingivitis in both groups at week 2.

Example 22

The same clinical samples from experimental gingivitis (EXAMPLE 20) were analyzed using the procedures described in EXAMPLE 17. The bound ornithine was the lowest at week 0 (FIG. 22) in both groups. Its levels at the baseline were higher than those at week 0. The bound ornithine reached peaks when gingivitis was induced in both groups at week 2. Also it is worth noting the total ornithine (Free and protein bound ornithine) was increased in the induced gingivitis (FIG. 23) in both groups.

Example 23

The same procedures were used as described in EXAMPLE 17. The samples were the same as described in EXAMPLE 20. The protein bound arginine was the lowest in induced gingivitis (FIG. 24) in both groups. Its levels were higher in WK0 than at Baseline in both groups. The total arginine in the gingival brush samples displayed the same patterns as the protein bound one (FIG. 25).

Example 24

Citrulline was purchased from Sigma-Aldrich (St. Louis, Mo.). THP1-Dual™ cells were purchased from Invivogen (San Diego, Calif.). Cells were cultured following the manufacturer's instruction, as described in EXAMPLE 11. For treatment, 0.3 mM to 9 mM of citrulline were first added to the culture medium. Then, 300 ng/ml of P. gingivalis LPS 1690 were added 60 minutes later. After 24 hours of treatment, media was collected and analyzed for cytokine production using 9-plex kit (Meso Scale Diagnostics Rockville, Md.).

P. gingivalis LPS 1690 stimulated cytokine production, as shown in FIG. 26. Citrulline inhibited P. gingivalis LPS 1690 effects on proinflammatory cytokine production in a dose-dependent manner. Those cytokines include IL-6, TNF-α, IL-12p70, IL-10, IL-2, IFN-r and IL-1β.

Example 26

Growth of bacteria: Two bacteria, Bacterium A and Bacterium B, were cultured in Tryptic Soy Broth medium (Sigma-Aldrich, St. Louis, Mo.) at 37° C. with shaking at 200 rpm. The bacteria were harvested at 24 hours, and suspended in 0.5 ml of phosphate-buffered saline, labeled “live”. Half ml of “Live” bacteria was transferred to a 1.5 ml microtube, and heated to 80° C. for 30 min. The heat-treated bacteria were labeled “Heat-Inactivated”, or “Dead”.

Measurement of TLR responses in THP-1 gene reporter cells (NFkB-SEAP): The Live and Heat-Inactivated bacteria were applied to THP-1 cells as described in EXAMPLE 11. As shown in FIG. 31, EC50 of Bacterium A and B on activation of NFkB-SEAP reporter gene in THP-1 cells was determined. Both Live and Heat-inactivated (Dead) bacteria stimulated expression of the NFkB-SEAP reporter gene. Bacterium B had a lower EC50 than Bacterium A in activating expression of the NFkB-SEAP reporter gene.

Cytokine production and measurement: Human peripheral bleed mononuclear cells (hPBMC) were obtained from All Cells company (All Cells, Alameda, Calif.) as Leukapheresed blood. Leukapheresed blood was mixed with an equal part of DMEM+glutaGRO supplemented with 9.1% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher, Waltham, Mass.). hPBMC were isolated from the 1:1 mixture of blood and culture medium by collecting the buffy coat of a centrifuged Histopaque®-1077 (Sigma-Aldrich, St. Louis, Mo.) buffer density gradient. The cells (200,000 cells) were cultured in 200 μl of DMEM+glutaGRO supplemented with 9.1% fetal bovine serum and 1% penicillin/streptomycin, and treated with Live and Heat-Inactive bacteria (6,250,000 colony-forming units). The medium was harvested at 24 hours after adding the bacteria, and analyzed for proinflammatory cytokines in a kit following manufacturer's instruction (Meso Scale Diagnostics, Rockville, Md.).

As shown in TABLE 9, both live bacterium A and B stimulated production of cytokines in hPBMC. Bacteriun B was far more potent than Bacterium A in promoting production of IFN-γ, IL-10, IL-12p70, IL-1β, IL-6, IL-8 and TNF-α in hPBMC.

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.”

Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.