Study protocol for a pilot quasi-experimental study on oral health education for nurses and community health workers in Nigeria.

Introduction: The primary health care system provides an ideal setting for the integration of oral health into general health care as well as equitable access to oral health care. However, the limited oral health knowledge of primary health care workers necessitates appropriate training before they can participate in health promotion efforts. This pilot training was designed to examine the impact of the Oral Health Education module for Nurses and Community Health Care Workers on their oral health awareness and referral practices. Methods: This study will utilize a quasi-experimental design (pre-and post with a non-equivalent control group) to assess the impact of a five-day pilot oral health education program on the knowledge and referral practices of Nurses and Community Health Workers in primary health care centers in three states in Nigeria-(Lagos, Oyo, and Kano). The training modules were developed based on the six iterative steps described in the intervention mapping framework - needs assessment, highlighting program objectives and outcomes, selection of theory and mode of intervention, designing program based on theory, designing implementation plans, and developing an evaluation plan. Only the intervention group will participate in the full educational training sessions but both groups will complete the pre-and post-intervention questionnaires. Discussion: This pilot training combined the standardized training modules from the recently launched "Oral Health Training Course for Community Health Workers in Africa" and a newly developed maternal and child oral health module by our group using an evidence-based approach. To the best of our knowledge, this is the first program to examine the impact of the standardized OpenWHO modules. The success of this training will lay the foundation for developing a sustained channel for providing oral health education at the primary health care level in Nigeria, West Africa, and Africa.

Enhancement of innate immunity in gingival epithelial cells by vitamin D and HDAC inhibitors.

Introduction: The human host defense peptide LL-37 is a component of the innate immune defense mechanisms of the oral cavity against colonization by microbes associated with periodontal disease. We have previously shown that the active form of vitamin D, 1,25(OH)2D3, can induce the expression of LL-37 in gingival epithelial cells (GEC), and prevent the invasion and growth of periopathogenic bacteria in these cells. Further, experimental vitamin D deficiency resulted in increased gingival inflammation and alveolar bone loss. Epidemiological studies have shown associations between vitamin D deficiency and periodontal disease in humans, suggesting application of vitamin D could be a useful therapeutic approach. Further, since we have shown the local activation of vitamin D by enzymes expressed in the GEC, we hypothesized that we could observe this enhancement with the stable, and inexpensive inactive form of vitamin D, which could be further increased with epigenetic regulators. Methods: We treated 3-dimensional primary cultures of GEC topically with the inactive form of vitamin D, in the presence and absence of selected histone deacetylase (HDAC) inhibitors. LL-37 mRNA levels were quantified by quantitative RT-PCR, and inhibition of invasion of bacteria was measured by fluorescence microscopy. Results: Vitamin D treatment led to an induction of LL-37 mRNA levels, as well as an inhibition of pro-inflammatory cytokine secretion. This effect was further enhanced by HDAC inhibitors, most strongly when the HDAC inhibitor, phenyl butyrate (PBA) was combined with Vitamin D3. This was observed both in solution and in a prototype gel formulation using sodium butyrate. Finally, this combination treatment led to an increase in the antimicrobial activity against infection by Porphyromonas gingivalis and Filifactor alocis, bacteria associated with periodontal lesions, as well as herpes simplex virus, which has also been shown to be associated with periodontal lesions. Conclusions: Our results demonstrate that a combination of inactive vitamin D and sodium butyrate could be developed as a safe treatment for periodontal disease.

Oral Epithelial Cells Expressing Low or Undetectable Levels of Human Angiotensin-Converting Enzyme 2 Are Susceptible to SARS-CoV-2 Virus Infection In Vitro.

The oral cavity is thought to be one of the portals for SARS-CoV-2 entry, although there is limited evidence of active oral infection by SARS-CoV-2 viruses. We assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication-competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Distinct viral dynamics were seen in hTERT TIGKs compared to A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells on day 3 after infection. Analysis of oral epithelial cells infected by replication-competent SARS-CoV-2 viruses expressing GFP showed that the GFP signal and SARS-CoV-2 mRNAs were not evenly distributed. Furthermore, we found cumulative SARS-CoV-2 RNAs from released viruses in the media from oral epithelial cells on day 1 and day 2 after infection, indicating productive viral infection. Taken together, our results demonstrated that oral epithelial cells were susceptible to SARS-CoV-2 viruses despite low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for the development of future vaccines and therapeutics.

Arginine Induced Streptococcus gordonii Biofilm Detachment Using a Novel Rotating-Disc Rheometry Method.

Oral diseases are one of the most common pathologies affecting human health. These diseases are typically associated with dental plaque-biofilms, through either build-up of the biofilm or dysbiosis of the microbial community. Arginine can disrupt dental plaque-biofilms, and maintain plaque homeostasis, making it an ideal therapeutic to combat the development of oral disease. Despite our understanding of the actions of arginine towards dental plaque-biofilms, it is still unclear how or if arginine effects the mechanical integrity of the dental plaque-biofilm. Here we adapted a rotating-disc rheometry assay, a method used to quantify marine biofilm fouling, to study how arginine treatment of Streptococcus gordonii biofilms influences biofilm detachment from surfaces. We demonstrate that the assay is highly sensitive at quantifying the presence of biofilm and the detachment or rearrangement of the biofilm structure as a function of shear stress. We demonstrate that arginine treatment leads to earlier detachment of the biofilm, indicating that arginine treatment weakens the biofilm, making it more susceptible to removal by shear stresses. Finally, we demonstrate that the biofilm disrupting affect is specific to arginine, and not a general property of amino acids, as S. gordonii biofilms treated with either glycine or lysine had mechanical properties similar to untreated biofilms. Our results add to the understanding that arginine targets biofilms by multifaceted mechanisms, both metabolic and physical, further promoting the potential of arginine as an active compound in dentifrices to maintain oral health.

Salivary SARS-CoV-2 load reduction with mouthwash use: A randomized pilot clinical trial.

The saliva of patients with COVID-19 has a high SARS-CoV-2 viral load. The risk of spreading the virus is high, and procedures for viral load reduction in the oral cavity are important. Little research to date has been performed on the effect of mouthwashes on the salivary SARS-CoV-2 viral load. This pilot randomized single-center clinical trial investigated whether three types of mouthwash with solutions containing either 0.075% cetylpyridinium chloride plus 0.28% zinc lactate (CPC + Zn), 1.5% hydrogen peroxide (HP), or 0.12% chlorhexidine gluconate (CHX) reduce the SARS-CoV-2 viral load in saliva at different time points. Sixty SARS-CoV-2-positive patients were recruited and randomly partitioned into a placebo (oral rinsing with distilled water) group and other groups according to the type of mouthwash. Saliva samples were collected from the participants before rinsing (T0), immediately after rinsing (T1), 30 min after rinsing (T2), and 60 min after rinsing (T3). The salivary SARS-CoV-2 viral load was measured by qRT-PCR assays. Rinsing with HP and CPC + Zn resulted in better reductions in viral load, with 15.8 ± 0.08- and 20.4 ± 3.7-fold reductions at T1, respectively. Although the CPC + Zn group maintained a 2.6 ± 0.1-fold reduction at T3, this trend was not observed for HP. HP mouthwash resulted in a significant reduction in the SARS-CoV-2 viral load up to 30 min after rinsing (6.5 ± 3.4). The CHX mouthwash significantly reduced the viral load at T1, T2, and T3 (2.1 ± 1.5-, 6.2 ± 3.8-, and 4.2 ± 2.4-fold reductions, respectively). In conclusion, mouthwash with CPC + Zinc and CHX resulted in significant reductions of the SARS-CoV-2 viral load in saliva up to 60 mins after rinsing, while HP mouthwash resulted in a significant reduction up to 30 mins after rinsing. Despite this transitory effect, these results encourage further studies and suggest that these products could be considered as risk-mitigation strategies for patients infected with SARS-CoV-2.

Enhanced In Vitro Zinc Bioavailability through Rational Design of a Dual Zinc plus Arginine Dentifrice.

OBJECTIVES: To investigate bioavailability enhancement of zinc on model oral surfaces and in oral biofilms in vitro through strategic formulation with two sources of zinc and L-arginine. METHODS: To modulate the bioavailability of active zinc ions in a zinc citrate dentifrice, an additive research strategy was pursued. A series of zinc citrate dentifrice formulations were prepared with increasing replacement of zinc citrate with zinc oxide (a water insoluble source of zinc ions) to generate a Dual Zinc active system. A screening of isolated zinc and amino acid effects in simple solutions using zeta potential and uptake to model oral surfaces was performed in an effort to determine the effect of particle charge on zinc bioavailability. Zinc delivery and antibacterial efficacy of the Dual Zinc plus Arginine dentifrice formula were tested using in vitro oral epithelial tissue and saliva-derived biofilm models. Furthermore, zinc penetration and retention were determined by subjecting in vitro biofilms to dynamic flow after treatment with the Dual Zinc plus Arginine dentifrice with treated biofilms evaluated for zinc using imaging mass spectrometry (I-MS). Bacterial adhesion to gingival epithelial cells treated with the Dual Zinc plus Arginine dentifrice was imaged upon challenging with Streptococcus gordonii. RESULTS: Addition of zinc oxide into a zinc citrate dentifrice formula enhanced the efficacy of the system against anaerobic biofilms in a concentration- dependent manner. L-arginine further provided a significant positive charge (+36 mV) to the zinc oxide suspension (+16 mV) as measured by zeta potential. Simple solutions of the Dual Zinc active showed increased zinc uptake on model oral surfaces as a direct function of L-arginine concentration. Antibacterial efficacy of a Dual Zinc plus Arginine dentifrice was evaluated through multiple mechanisms. Enhanced antibacterial performance was observed through significant reductions in metabolic activity as measured through bacterial glycolytic function (p = 0.0001) and total oxygen consumption (p = 0.0001). Greater penetration and retention of zinc was observed in bacterial biofilms treated with the Dual Zinc plus Arginine dentifrice in comparison to treatment with a Dual Zinc dentifrice after twelve hours of dynamic flow (10 mL/hour) in an in vitro drip flow biofilm culture. Confocal microscopy showed adherent bacteria on cheek cells treated with the Dual Zinc plus Arginine dentifrice formula. CONCLUSIONS: The combination of zinc citrate, zinc oxide, and the amino acid L-arginine in a dentifrice formula enhances the bioavailability of zinc to model oral tissue surfaces, resulting in unique physicochemical effects. The significant antimicrobial control associated with the Dual Zinc plus Arginine dentifrice provides a unique vehicle toward achieving whole mouth health.

Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus.

The flaviviruses dengue, West Nile, and Japanese encephalitis represent three major mosquito-borne viruses worldwide. These pathogens impact the lives of millions of individuals and potentially could affect non-endemic areas already colonized by mosquito vectors. Unintentional transport of infected vectors (Aedes and Culex spp.), traveling within endemic areas, rapid adaptation of the insects into new geographic locations, climate change, and lack of medical surveillance have greatly contributed to the increase in flaviviral infections worldwide. The mechanisms by which flaviviruses alter the immune and the central nervous system have only recently been examined despite the alarming number of infections, related deaths, and increasing global distribution. In this review, we will discuss the expansion of the geographic areas affected by flaviviruses, the potential threats to previously unaffected countries, the mechanisms of pathogenesis, and the potential therapeutic interventions to limit the devastating consequences of these viruses.

Structural dissection and in vivo effectiveness of a peptide inhibitor of Porphyromonas gingivalis adherence to Streptococcus gordonii.

The interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm by Porphyromonas gingivalis. We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interaction in vitro. Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reduces P. gingivalis colonization and alveolar bone loss in vivo in a murine model of periodontitis. Substitution of Gln for Pro(1171) or Glu(1168) increased the α-helicity of BAR and reduced its inhibitory activity in vitro by 10-fold and 2-fold, respectively. To determine if BAR prevents P. gingivalis infection in vivo, mice were first infected with Streptococcus gordonii and then challenged with P. gingivalis in the absence and presence of BAR. Animals that were infected with either 10(9) CFU of S. gordonii DL-1 or 10(7) CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10(9) CFU of S. gordonii followed by 10(7) CFU of P. gingivalis induced significantly greater bone loss (P < 0.01) than sham infection or infection of mice with either organism alone. S. gordonii-infected mice that were subsequently challenged with 10(7) CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity by P. gingivalis.

Selective substitution of amino acids limits proteolytic cleavage and improves the bioactivity of an anti-biofilm peptide that targets the periodontal pathogen, Porphyromonas gingivalis.

The interaction of the periodontal pathogen, Porphyromonas gingivalis, with oral streptococci such as Streptococcus gordonii precedes colonization of the subgingival pocket and represents a target for limiting P. gingivalis colonization of the oral cavity. Previous studies showed that a synthetic peptide (designated BAR) derived from the antigen I/II protein of S. gordonii was a potent competitive inhibitor of P. gingivalis adherence to S. gordonii and subsequent biofilm formation. Here we show that despite its inhibitory activity, BAR is rapidly degraded by intact P. gingivalis cells in vitro. However, in the presence of soluble Mfa protein, the P. gingivalis receptor for BAR, the peptide is protected from proteolytic degradation suggesting that the affinity of BAR for Mfa is higher than for P. gingivalis proteases. The rate of BAR degradation was reduced when the P. gingivalis lysine-specific gingipain was inhibited using the specific protease inhibitor, z-FKcK, or when the gene encoding the Lys-gingipain was inactivated. In addition, substituting d-Lys for l-Lys residues in BAR prevented degradation of the peptide when incubated with the Lys-gingipain and increased its specific adherence inhibitory activity in a S. gordonii-P. gingivalis dual species biofilm model. These results suggest that Lys-gingipain accounts in large part for P. gingivalis-mediated degradation of BAR and that more effective peptide inhibitors of P. gingivalis adherence to streptococci can be produced by introducing modifications that limit the susceptibility of BAR to the Lys-gingipain and other P. gingivalis associated proteases.

Tobacco upregulates P. gingivalis fimbrial proteins which induce TLR2 hyposensitivity.

BACKGROUND: Tobacco smokers are more susceptible to periodontitis than non-smokers but exhibit reduced signs of clinical inflammation. The underlying mechanisms are unknown. We have previously shown that cigarette smoke extract (CSE) represents an environmental stress to which P. gingivalis adapts by altering the expression of several virulence factors - including major and minor fimbrial antigens (FimA and Mfa1, respectively) and capsule - concomitant with a reduced pro-inflammatory potential of intact P. gingivalis. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that CSE-regulation of capsule and fimbrial genes is reflected at the ultrastructural and functional levels, alters the nature of host-pathogen interactions, and contributes to the reduced pro- inflammatory potential of smoke exposed P. gingivalis. CSE induced ultrastructural alterations were determined by electron microscopy, confirmed by Western blot and physiological consequences studied in open-flow biofilms. Inflammatory profiling of specific CSE-dysregulated proteins, rFimA and rMfa1, was determined by quantifying cytokine induction in primary human innate and OBA-9 cells. CSE up-regulates P. gingivalis FimA at the protein level, suppresses the production of capsular polysaccharides at the ultrastructural level, and creates conditions that promote biofilm formation. We further show that while FimA is recognized by TLR2/6, it has only minimal inflammatory activity in several cell types. Furthermore, FimA stimulation chronically abrogates the pro-inflammatory response to subsequent TLR2 stimulation by other TLR-2-specific agonists (Pam3CSK4, FSL, Mfa1) in an IkappaBalpha- and IRAK-1-dependent manner. CONCLUSIONS/SIGNIFICANCE: These studies provide some of the first information to explain, mechanistically, how tobacco smoke changes the P. gingivalis phenotype in a manner likely to promote P. gingivalis colonization and infection while simultaneously reducing the host response to this major mucosal pathogen.

Autoinducer-2 and QseC control biofilm formation and in vivo virulence of Aggregatibacter actinomycetemcomitans.

Biofilm formation by the periodontal pathogen Aggregatibacter actinomycetemcomitans is dependent upon autoinducer-2 (AI-2)-mediated quorum sensing. However, the components that link the detection of the AI-2 signal to downstream gene expression have not been determined. One potential regulator is the QseBC two-component system, which is part of the AI-2-dependent response pathway that controls biofilm formation in Escherichia coli. Here we show that the expression of QseBC in A. actinomycetemcomitans is induced by AI-2 and that induction requires the AI-2 receptors, LsrB and/or RbsB. Additionally, inactivation of qseC resulted in reduced biofilm growth. Since the ability to grow in biofilms is essential for A. actinomycetemcomitans virulence, strains that were deficient in QseC or the AI-2 receptors were examined in an in vivo mouse model of periodontitis. The DeltaqseC mutant induced significantly less alveolar bone resorption than the wild-type strain (P < 0.02). Bone loss in animals infected with the DeltaqseC strain was similar to that in sham-infected animals. The DeltalsrB, DeltarbsB, and DeltalsrB DeltarbsB strains also induced significantly less alveolar bone resorption than the wild type (P < 0.03, P < 0.02, and P < 0.01, respectively). However, bone loss induced by a DeltaluxS strain was indistinguishable from that induced by the wild type, suggesting that AI-2 produced by indigenous microflora in the murine oral cavity may complement the DeltaluxS mutation. Together, these results suggest that the QseBC two-component system is part of the AI-2 regulon and may link the detection of AI-2 to the regulation of downstream cellular processes that are involved in biofilm formation and virulence of A. actinomycetemcomitans.

Interaction of Porphyromonas gingivalis with oral streptococci requires a motif that resembles the eukaryotic nuclear receptor box protein-protein interaction domain.

Porphyromonas gingivalis initially colonizes the oral cavity by interacting with organisms in supragingival plaque, such as the oralis group of oral streptococci. This interaction involves the association of the streptococcal antigen I/II with the minor fimbrial antigen (Mfa1) of P. gingivalis. Our previous studies showed that a peptide (BAR) derived from antigen I/II inhibits P. gingivalis adherence and subsequent biofilm formation on streptococcal substrates. In addition, screening a combinatorial peptide library identified select amino acid substitutions in the NITVK active region of BAR that increased the adherence of P. gingivalis to streptococci. Here we report that incorporating these residues in a synthetic peptide results in more-potent inhibition of P. gingivalis adherence and biofilm formation (I(50) [50% inhibition] at 0.52 microM versus I(50) at 1.25 microM for BAR). In addition, a second structural motif in BAR, comprised of the amino acids KKVQDLLKK, was shown to contribute to P. gingivalis adherence to streptococci. Consistent with this, the KKVQDLLKK and NITVK motifs are conserved only in antigen I/II proteins expressed by the oralis group of streptococci, which interact with P. gingivalis. Interestingly, the primary and secondary structures and the functional characteristics of the amphipathic VQDLL core alpha-helix resemble the consensus nuclear receptor (NR) box protein-protein interacting domain sequence (LXXLL) of eukaryotes. BAR peptides containing amino acid substitutions with the potential to disrupt the secondary structure of VQDLL were less-effective inhibitors of P. gingivalis adherence and biofilm formation, suggesting that the alpha-helical character of VQDLL is important. Furthermore, replacing the lysines that flank VQDLL with acidic amino acids also reduced inhibitory activity, suggesting that the association of VQDLL with Mfa1 may be stabilized by a charge clamp. These results indicate that the Mfa1-interacting interface of streptococcal antigen I/II encompasses both the KKVQDLLKK and NITVK motif and suggest that the adherence of P. gingivalis to streptococci is driven by a protein-protein interaction domain that resembles the eukaryotic NR box. Thus, both motifs must be taken into account in designing potential peptidomimetics that target P. gingivalis adherence and biofilm formation.

Structural characterization of peptide-mediated inhibition of Porphyromonas gingivalis biofilm formation.

Porphyromonas gingivalis is a periodontal pathogen whose primary niche is the anaerobic environment of subgingival dental plaque, but initial colonization of the oral cavity is likely to occur on supragingival surfaces that already support robust biofilm communities. Our studies have shown that P. gingivalis adheres to Streptococcus gordonii through interaction of the minor fimbrial antigen Mfa1 with a specific region of the streptococcal SspB polypeptide (residues 1167 to 1193) designated BAR. We show that a synthetic peptide comprising the BAR sequence potently inhibits P. gingivalis adherence to S. gordonii (50% inhibitory concentration = 1.3 microM) and prevents the development of P. gingivalis biofilms. However, a retroinverso peptide that possessed the same side chain topology as that of BAR was inactive, suggesting that interactions of Mfa1 with the peptide backbone of BAR are important for binding. A conformationally constrained analog of BAR inhibited P. gingivalis adherence and biofilm formation but at a lower specific activity than that of BAR. Therefore, to further define the structural features of the Mfa1-BAR interaction, we functionally screened combinatorial libraries of BAR in which active site residues (Asn1182, Thr1184, and Val1185) were replaced with each of the 19 common amino acids. Peptides containing positively charged amino acids at position 1182 or hydrophobic residues at position 1185 bound P. gingivalis more efficiently than did control peptides containing Asn and Val at these positions, suggesting that electrostatic and hydrophobic interactions may contribute to Mfa1-SspB binding. In contrast, replacement of Pro or Gly at these positions was detrimental to adherence, suggesting that perturbation of the BAR secondary structure influences activity. The net effect of substitutions for Thr1184 was less pronounced either positively or negatively than that at the other sites. These results define physicochemical characteristics of the interacting interface of Mfa1 and SspB and suggest that peptides or peptidomimetics with greater specific inhibitory activity than that of BAR can be developed. These compounds may represent potential therapeutics that target some of the first molecular interactions that allow P. gingivalis to colonize the oral cavity.