Unexpected lower level of oral periodontal pathogens in patients with high numbers of systemic diseases.

Background: Periodontal disease is associated with systemic conditions such as diabetes, arthritis, and cardiovascular disease, all diseases with large inflammatory components. Some, but not all, reports show periopathogens Porphyromonas gingivialis and Tannerella forsythia at higher levels orally in people with one of these chronic diseases and in people with more severe cases. These oral pathogens are thought to be positively associated with systemic inflammatory diseases through induction of oral inflammation that works to distort systemic inflammation or by directly inducing inflammation at distal sites in the body. This study aimed to determine if, among patients with severe periodontal disease, those with multi-morbidity (or many chronic diseases) showed higher levels of periodontal pathogens. Methods: A total of 201 adult subjects, including 84 with severe periodontal disease were recruited between 1/2017 and 6/2019 at a city dental clinic. Electronic charts supplied self-reported diseases and conditions which informed a morbidity index based on the number of chronic diseases and conditions present. Salivary composition was determined by 16S rRNA gene sequencing. Results: As expected, patients with severe periodontal disease showed higher levels of periodontal pathogens in their saliva. Also, those with severe periodontal disease showed higher levels of multiple chronic diseases (multimorbidity). An examination of the 84 patients with severe periodontal disease revealed some subjects despite being of advanced age were free or nearly free of systemic disease. Surprisingly, the salivary microbiota of the least healthy of these 84 subjects, defined here as those with maximal multimorbidity, showed significantly lower relative numbers of periodontal pathogens, including Porphyromonas gingivalis and Tannerella Forsythia, after controlling for active caries, tobacco usage, age, and gender. Analysis of a control group with none to moderate periodontal disease revealed no association of multimorbidity or numbers of medications used and specific oral bacteria, indicating the importance of severe periodontal disease as a variable of interest. Conclusion: The hypothesis that periodontal disease patients with higher levels of multimorbidity would have higher levels of oral periodontal pathogens is false. Multimorbidity is associated with a reduced relative number of periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia.

Oral microbial taxa associated with risk for SARS-CoV-2 infection.

Hypothesis and objective: The oral and digestive tract microbial ecosystem has sparked interest because of its impact on various systemic diseases and conditions. The oral cavity serves not only as a reservoir for many potentially virulent microbiota but also as an important entry point and portal to the human body system. This is especially significant in the transmissibility of the virulent current pandemic virus SARS-CoV-2. The oral and digestive microbiome influences the inflammatory burden and effectiveness of the immune system and serves as a marker of activity of these host processes. The host immune response plays a role in infection susceptibility, including SARS-CoV-2. The purpose of this study is to investigate the role of specific salivary oral microbiome in susceptibility to SARS-CoV-2 infection. Methods and results: One hundred six subjects of known medical and dental history who consented to provide saliva samples between January 2017 and December 2019 were included in this study. Sixteen had become COVID-19 positive based on the PCR test by 3/01/2021. A comparison of oral microbiome bacteria taxa profiles based on 16S rRNA sequencing revealed differences between the two groups in this pilot study. Conclusions: These bacteria taxa may be markers of increased susceptibility to SARS-CoV-2 infection in the unvaccinated population.

Old age and other factors associated with salivary microbiome variation.

BACKGROUND: Many factors can contribute to the exact makeup of the salivary microbiome. Differences in the oral microbiome occur with old age, which may be due to oral conditions and diseases associated with old age, such as edentulism, as well as other unknown causes. METHODS: The salivary microbiome was sampled in patients from a large urban clinic. For all subjects age, gender, periodontal status, caries status, presence of edentulism, medications, and tobacco usage were recorded. Multifactor analysis was used to study variation in salivary microbiome profiles linked to these factors. RESULTS: In the population sampled, there were significantly higher numbers of edentulous subjects, and increased levels of polypharmacy found with aging. Large differences in alpha diversity and beta diversity of the salivary microbiome in the old age group were largely linked to edentulism. However, multivariable analysis revealed, even after adjusting for differences in edentulism, polypharmacy, tobacco usage, periodontal disease, caries level, and gender, that old age itself was associated with lower levels of taxa Porphyromonas endodontalis, Alloprevotella tannerae, Filifactor alocis, Treponema, Lautropia Mirabilis and Pseudopropionibacterium sp._HMT_194. Surprisingly, of these taxa, most were ones known to reside on or near tooth surfaces. CONCLUSIONS: Another factor or factors beyond edentulism, polypharmacy and periodontal disease play a role in the differences seen in oral microbiome with old age. The nature of this factor(s) is not known.

Comparison of Microbiome in Stimulated Saliva in Edentulous and Dentate Subjects.

Measurement of saliva microbes is promoted as a way to detect oral and systemic disease, yet there is a multitude of factors that affect the oral microbiome. The salivary microbiome is influenced by biofilm of shedding (epithelial) and non-shedding (tooth) surfaces. Methods for study of the salivary microbiome are by no means standardized, and differences in sample collection, storage, and processing can all affect results to some degree. Here we describe one method of saliva collection that has been validated for reproducibility. Standard 16S rRNA gene analysis is done using the Human Oral Microbiome Database library which results in analysis that is straightforward. Everything about this procedure except the library synthesis and DNA sequencing itself can easily be done in-house. To gauge the ability of salivary microbial analytics to distinguish between edentulous and dentate oral conditions, differences in the saliva microbiome of subjects with and without teeth were examined. Fifty-two dentate and 49 edentulous subjects provided stimulated saliva samples. 16S rRNA gene sequencing, QIIME-based data processing, and statistical analysis were done using several different analytical approaches to detect differences in the salivary microbiome between the two groups. Bacteria diversity was lower in the edentulous group. Remarkably, all 31 of the most significant differences in taxa were deficits that occurred in the edentulous group. As one might expect, many of these taxa are attributed to dental plaque and gingival sulcus-associated bacteria verifying that the measurement of 16S rRNA genes in the bacteria of the saliva can be used to reproducibly measure expected differences in the oral microbiome that occur with edentulism or other conditions and diseases.

Noninvasive Acquisition of Oral Mucosal Epithelial miRNA and Bacteria DNA/RNA from a Single Site.

Many digestive tract microbes live adhered to tract epithelium. Work in recent years has brought the realization that these microbes and the host epithelial cells certainly must interact and that this interaction has an effect on both. One way to understand the interaction is to measure which genes are expressed in the epithelial cells and what bacteria are present. Even more informative would be to also determine what genes the bacteria express. Presented is a method to noninvasively isolate oral mucosal epithelium so to provide purified miRNA that can be used to profile miRNA expression specifically in the epithelium. miRNA is a major regulator of cell functions. Simultaneously, DNA and RNA from bacteria at the same site can be isolated to allow characterization of bacteria that coat the epithelial cells and extracellular matrix. This provides insight on the interaction between host and bacteria.

Salivary microbiome with gastroesophageal reflux disease and treatment.

The effect of oral microbial composition on periodontal health and on systemic health has been, and is being established. The oral microbiome, in turn, can be altered by local and systemic diseases and conditions. Gastroesophageal reflux disease (GERD), has been associated with increased acidity in the oral cavity resulting in dental erosion, and controversially a reduced risk of periodontal disease. We hypothesized that presence of GERD was linked to a modified microbial profile in untreated GERD patients and that the use of proton pump inhibitor (PPI) drugs: potent disruptors of gut microbiome, in GERD patients might result in a salivary microbiome that is further distinct. Untreated GERD patients showed multiple differences in salivary microbiome as compared to healthy controls. Taxa found at lower levels related to the presence of GERD not treated by PPI included: Prevotella melaninogenica, Prevotella pallens, Leptotrichia, and Solobacterium moorei and thirteen others. In contrast, GERD patients chronically using PPI showed minimal differences in salivary taxa compared to healthy controls not using PPI.

Gut/Oral Bacteria Variability May Explain the High Efficacy of Green Tea in Rodent Tumor Inhibition and Its Absence in Humans.

Consumption of green tea (GT) and GT polyphenols has prevented a range of cancers in rodents but has had mixed results in humans. Human subjects who drank GT for weeks showed changes in oral microbiome. However, GT-induced changes in RNA in oral epithelium were subject-specific, suggesting GT-induced changes of the oral epithelium occurred but differed across individuals. In contrast, studies in rodents consuming GT polyphenols revealed obvious changes in epithelial gene expression. GT polyphenols are poorly absorbed by digestive tract epithelium. Their metabolism by gut/oral microbial enzymes occurs and can alter absorption and function of these molecules and thus their bioactivity. This might explain the overall lack of consistency in oral epithelium RNA expression changes seen in human subjects who consumed GT. Each human has different gut/oral microbiomes, so they may have different levels of polyphenol-metabolizing bacteria. We speculate the similar gut/oral microbiomes in, for example, mice housed together are responsible for the minimal variance observed in tissue GT responses within a study. The consistency of the tissue response to GT within a rodent study eases the selection of a dose level that affects tumor rates. This leads to the theory that determination of optimal GT doses in a human requires knowledge about the gut/oral microbiome in that human.

Deep DNA metagenomic sequencing reveals oral microbiome divergence between monozygotic twins discordant for multiple sclerosis severity.

In contrast to gut, the oral microbiome of MS patients has not been characterized. Deep sequencing of saliva DNA from a pair of monozygotic twins (MSF1 with relapsing remitting MS; MSF2 with clinically isolated syndrome) identified 2036 bacterial species. Relative abundances of 3 phyla were higher, and 3 lower in MSF1 versus MSF2. Species diversity was greater in MSF2, and 20 abundant species differed at least 2-fold. Pathway analysis identified 116 functional hierarchies differing 50% or more. Although limited to one pair of twins, our data suggests that oral microbiome analysis may be useful for diagnosis or monitoring therapeutic efficacy.

Microbiomic differences at cancer-prone oral mucosa sites with marijuana usage.

Marijuana smoke contains cannabinoids, immunosuppressants, and a mixture of potentially-mutagenic chemicals. In addition to systemic disease, it is thought to contribute to oral disease, such as tooth loss, tissue changes in the gums and throat, and possibly oral pharyngeal cancer. We used a cross-sectional study of 20 marijuana users and 19 control non-users, to determine if chronic inhalation-based exposure to marijuana was associated with a distinct oral microbiota at the two most common sites of head and neck squamous cell carcinoma (HNSCC), the lateral border of the tongue and the oral pharynx. At the tongue site, genera earlier shown to be enriched on HNSCC mucosa, Capnocytophaga, Fusobacterium, and Porphyromonas, were at low levels in marijuana users, while Rothia, which is found at depressed levels on HNSCC mucosa, was high. At the oral pharynx site, differences in bacteria were distinct, with higher levels of Selenomonas and lower levels of Streptococcus which is what is seen in HNSCC. No evidence was seen for a contribution of marijuana product contaminating bacteria to these differences. This study revealed differences in the surface oral mucosal microbiota with frequent smoking of marijuana.

A subtype of oral, laryngeal, esophageal, and lung, squamous cell carcinoma with high levels of TrkB-T1 neurotrophin receptor mRNA.

BACKGROUND: The NTRK2 genetic locus encodes neurotrophin membrane receptors that play an important role in normal neural tissue plasticity, growth, and survival. One NTRK2-encoded protein is TrkB-FL, which can regulate multiple pathways relevant to cancer. A second NTRK2 gene mRNA isoform encodes TrkB-T1, a receptor that has a different cytoplasmic domain encoded in a mRNA with a unique 3' terminal exon. METHOD: Tumors from The Cancer Genome Atlas (TCGA) and other studies were classified according to the expression of a single form of NTRK2 mRNA, TrkB-T1, identified by its unique 3' terminal exon. Analysis of differentially expressed genes in TrkB-T1 high expressers was done to determine if tumors enriched for TrkB-T1 mRNA were a uniform group independent of anatomic site. RESULTS: The mRNA for TrkB-T1 is the most abundant NTRK2 gene mRNA in all squamous cell carcinomas (SCCs) in the TCGA database. Comparison of larynx SCC high TrkB-T1 RNA expressers to low expressers (n = 96) revealed gene expression differences consistent with the high TrkB-T1 tumors being more neural-like. The upregulated genes in the TrkB-T1 RNA high expressers also showed enrichment of pathways involved in retinol metabolism, hedgehog signaling, and the Nfe2l2 response, among other pathways. An examination of oral, esophagus, and lung SCCs (n = 284, 97, 501) showed induction of the same pathways among tumors that expressed high levels of TrkB-T1 mRNA. Proteins associated with regulation of the sonic hedgehog pathway, and the Nfe2l2 response, Tp63, and Keap1 and p62/SQSTM1 proteins, showed differential expression in larynx, oral and lung high TrkB1-T1 expresser SCCs. Unexpectantly, the relationship of high level TrkB-T1 expression to patient outcomes was SCC anatomic site specific. High TrkB-T1 mRNA levels in laryngeal SCC correlated with poor survival, but the opposite was true for lung SCC. This may be because pathways enriched in the TrkB high expressers, like those involving oncogenes NFE2L2, PIK3CA, and SOX2, are known to have SCC anatomic site-specific effects on progression. CONCLUSIONS: High level TrkB-T1 mRNA is a marker of a distinct SCC subtype enriched for at least 3 pathways relevant to tumor progression: Nfe2l2 response, retinol metabolism, and hedgehog signaling.

Diversity of the oral microbiome between dentate and edentulous individuals.

BACKGROUND: Measurement of saliva microbes is promoted as a way to detect oral and systemic disease, yet there is a multitude of factors that affect the oral microbiome. The salivary microbiome is influenced by oral biofilm of shedding (epithelial) and non-shedding (tooth) surfaces. METHODS: To gauge the ability of salivary microbial analytics to distinguish between edentulous and dentate oral conditions, we looked for differences in the saliva microbiome of subjects with and without teeth. Fifty-two dentate and 49 edentulous subjects provided stimulated saliva samples. 16S rRNA gene sequencing, QIIME-based data processing, and statistical analysis were done using several different analytical approaches to detect differences in the salivary microbiome between the two groups. RESULTS: Bacteria diversity was lower in the edentulous group. Remarkably, all 31 of the most significant differences in taxa were deficits that occur in the edentulous group. As one might expect many of these taxa are attributed to dental plaque and gingival sulcus associated bacteria. CONCLUSION: In sum, the measurement of 16S rRNA genes in the bacteria of the saliva can be used to reproducibly measure differences in the oral microbiome that occur with edentulism, mainly the lack of tooth and tooth-related structures.

Effects of green tea on miRNA and microbiome of oral epithelium.

Consumption of green tea (GT) extracts or purified catechins has shown the ability to prevent oral and other cancers and inhibit cancer progression in rodent models, but the evidence for this in humans is mixed. Working with humans, we sought to understand the source of variable responses to GT by examining its effects on oral epithelium. Lingual epithelial RNA and lingual and gingival microbiota were measured before and after 4 weeks of exposure in tobacco smokers, whom are at high risk of oral cancer. GT consumption had on average inconsistent effects on miRNA expression in the oral epithelium. Only analysis that examined paired miRNAs, showing changed and coordinated expression with GT exposure, provided evidence for a GT effect on miRNAs, identifying miRNAs co-expressed with two hubs, miR-181a-5p and 301a-3p. An examination of the microbiome on cancer prone lingual mucosa, in contrast, showed clear shifts in the relative abundance of Streptococcus and Staphylococcus, and other genera after GT exposure. These data support the idea that tea consumption can consistently change oral bacteria in humans, which may affect carcinogenesis, but argue that GT effects on oral epithelial miRNA expression in humans vary between individuals.

Improving accuracy of RNA-based diagnosis and prognosis of oral cancer by using noninvasive methods.

RNA-based diagnosis and prognosis of squamous cell carcinoma has been slow to come to the clinic. Improvements in RNA measurement, statistical evaluation, and sample preservation, along with increased sample numbers, have not made these methods reproducible enough to be used clinically. We propose that, in the case of squamous cell carcinoma of the oral cavity, a chief source of variability is sample dissection, which leads to variable amounts of stroma mixed in with tumor epithelium. This heterogeneity of the samples, which requires great care to avoid, makes it difficult to see changes in RNA levels specific to tumor cells. An evaluation of the data suggests that, paradoxically, brush biopsy samples of oral lesions may provide a more reproducible method than surgical acquisition of samples for miRNA measurement. The evidence also indicates that body fluid samples can show similar changes in miRNAs with oral squamous cell carcinoma (OSCC) as those seen in tumor brush biopsy samples - suggesting much of the miRNA in these samples is coming from the same source: tumor epithelium. We conclude that brush biopsy or body fluid samples may be superior to surgical samples in allowing miRNA-based diagnosis and prognosis of OSCC in that they feature a rapid method to obtain homogeneous tumor cells and/or RNA.

microRNA from brush biopsy to characterize oral squamous cell carcinoma epithelium.

Few cancers are diagnosed based on RNA expression signatures. Oral squamous cell carcinoma (OSCC) is no exception; it is currently diagnosed by scalpel biopsy followed by histopathology. This study sought to identify oral tumor epithelial microRNA (miRNA) expression changes to determine if these changes could be used to diagnose the disease noninvasively. Analysis of miRNA profiles from surgically obtained OSCC tissue, collected under highly standardized conditions for The Cancer Genome Atlas, was done to determine the potential accuracy in differentiating tumor from normal mucosal tissue. Even when using small 20 subject datasets, classification based on miRNA was 90 to 100% accurate. To develop a noninvasive classifier for OSSC, analysis of brush biopsy miRNA was done and showed 87% accuracy in differentiating tumor from normal epithelium when using RT-qPCR or miRNAseq to measure miRNAs. An extensive overlap was seen in differentially expressed miRNAs in oral squamous cell carcinoma epithelium obtained using brush biopsy and those reported in saliva and serum of oral squamous cell carcinoma patients in several studies. This suggested that nonselective release of these miRNAs into body fluids from tumor epithelium was largely responsible for the changes in levels in these fluids seen with this disease. Using a variation in mirRPath we identified the KEGG pathway of neurotrophin signaling as a target of these miRNAs disregulated in tumor epithelium. This highlights the utility of brush biopsy of oral mucosa to allow simple acquisition of cancer relevant miRNA information from tumor epithelium.

A loss of profilin-1 in late-stage oral squamous cell carcinoma.

BACKGROUND: The genes for PFN1 and TMSB4 are both highly expressed in oral tissue and both encode actin monomer binding proteins thought to play a role in cell motility and possibly other crucial parts of tumor progression. METHODS: Oral brush cytology of epithelium from oral squamous cell carcinoma (OSCC) was used to measure PFN1 and TMSB4 mRNA in OSCC, while immunohistochemical analysis of tissue was used to check protein levels. RESULTS: High but variable expression of mRNAs encoding these two proteins was observed suggesting they may contribute to tumor characteristics in a subset of OSCCs. Both proteins were highly expressed in normal appearing basal epithelium, in the cytoplasm, and perinuclear area, while expression was minimal in upper epithelial layers. In OSCCs, expression of these proteins varied. In tumors classified as later stage, based on size and/or lymph node involvement, PFN1 levels were lower in tumor epithelium. A control gene, KRT13, showed expression in normal differentiated basal and suprabasal oral mucosa epithelial cells and as reported was lost in OSCC cells. CONCLUSION: Loss of PFN1 in tumor cells has been associated with lymph node invasion and metastasis in other tumor types, strengthening the argument that the protein has the potential to be a tumor suppressor in late-stage OSCC.

Similar Squamous Cell Carcinoma Epithelium microRNA Expression in Never Smokers and Ever Smokers.

The incidence of oral tumors in patients who never used mutagenic agents such as tobacco is increasing. In an effort to better understand these tumors we studied microRNA (miRNA) expression in tumor epithelium of never tobacco users, tumor epithelium of ever tobacco users, and nonpathological control oral epithelium. A comparison of levels among 372 miRNAs in 12 never tobacco users with oral squamous cell carcinoma (OSCC) versus 10 healthy controls was made using the reverse transcription quantitative polymerase chain reaction. A similar analysis was done with 8 ever tobacco users with OSCC. These comparisons revealed miR-10b-5p, miR-196a-5p, and miR-31-5p as enriched in the tumor epithelium in OSCC of both never and ever tobacco users. Examination of The Cancer Genome Atlas (TCGA) project miRNA data on 305 OSCCs and 30 controls revealed 100% of those miRNAs enriched in never smoker OSCCs in this patient group were also enriched in ever smoker OSCCs. Nonsupervised clustering of TCGA OSCCs was suggestive of two or four subgroups of tumors based on miRNA levels with limited evidence for differences in tobacco exposure among the groups. Results from both patient groups together stress the importance of miR196a-5p in OSCC malignancy in both never and ever smokers, and emphasize the overall similarity of miRNA expression in OSCCs in these two risk groups. It implies that there may be great similarity in etiology of OSCC in never and ever smokers and that classifying OSCC based on tobacco exposure may not be helpful in the clinic.

Gene expression based evidence of innate immune response activation in the epithelium with oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a disease of the oral mucosa of unknown cause producing lesions with an intense band-like inflammatory infiltrate of T cells to the subepithelium and keratinocyte cell death. We performed gene expression analysis of the oral epithelium of lesions in subjects with OLP and its sister disease, oral lichenoid reaction (OLR), in order to better understand the role of the keratinocytes in these diseases. DESIGN: Fourteen patients with OLP or OLR were included in the study, along with a control group of 23 subjects with a variety of oral diseases and a normal group of 17 subjects with no clinically visible mucosal abnormalities. Various proteins have been associated with OLP, based on detection of secreted proteins or changes in RNA levels in tissue samples consisting of epithelium, stroma, and immune cells. The mRNA level of twelve of these genes expressed in the epithelium was tested in the three groups. RESULTS: Four genes showed increased expression in the epithelium of OLP patients: CD14, CXCL1, IL8, and TLR1, and at least two of these proteins, TLR1 and CXCL1, were expressed at substantial levels in oral keratinocytes. CONCLUSIONS: Because of the large accumulation of T cells in lesions of OLP it has long been thought to be an adaptive immunity malfunction. We provide evidence that there is increased expression of innate immune genes in the epithelium with this illness, suggesting a role for this process in the disease and a possible target for treatment.

A prototype tobacco-associated oral squamous cell carcinoma classifier using RNA from brush cytology.

BACKGROUND: Oral cancer in the form of squamous cell carcinoma (OSCC) is typically detected in advanced stages when treatment is complex and may not be curative. The need for surgical biopsy may contribute to delays in diagnosis and impede early detection. Multiple studies of RNA from surgically obtained tumor samples have revealed many genes differentially expressed with this disease. We sought to determine whether the identified mRNAs could be used as markers by a non-invasive detection system for OSCC using RNA from brush cytology. METHODS: Levels of mRNAs from 21 genes known to be differentially expressed in head and neck squamous cell carcinoma surgical samples, compared with controls, were shown to be quantifiable in oral brush cytology samples. These mRNAs were quantified in a training set of 14 tumor and 20 non-malignant brush cytology samples from tobacco/betel nut users. With the measurement of two additional mRNAs and analysis using support vector machines algorithm for class prediction of these cancers was produced. RESULTS: This OSCC classifier based on the levels of 5 mRNAs in RNA from brush cytology initially showed 0.93 sensitivity and 0.91 specificity in differentiating OSCC from benign oral mucosal lesions based on leave-one-out cross-validation. When used on a test set of 19 samples from 6 OSCCs and 13 non-malignant oral lesions, we found misclassification of only one OSCC and one benign lesion. CONCLUSIONS: This shows the promise of using RNA from brush cytology for early OSCC detection and the potential for clinical usage of this non-invasive classifier.

Looking in the mouth for noninvasive gene expression-based methods to detect oral, oropharyngeal, and systemic cancer.

Noninvasive diagnosis, whether by sampling body fluids, body scans, or other technique, has the potential to simplify early cancer detection. A classic example is Pap smear screening, which has helped to reduce cervical cancer 75% over the last 50 years. No test is error-free; the real concern is sufficient accuracy combined with ease of use. This paper will discuss methods that measure gene expression or epigenetic markers in oral cells or saliva to diagnose oral and pharyngeal cancers, without requiring surgical biopsy. Evidence for lung and other distal cancer detection is also reviewed.