Single nucleotide polymorphisms conferring susceptibility to leukemia and oral mucositis: a multi-center pilot study of patients prior to conditioning therapy for hematopoietic cell transplant.

PURPOSE: Leukemias have been associated with oral manifestations, reflecting susceptibility to cancer therapy-induced oral mucositis. We sought to identify SNPs associated with both leukemia and oral mucositis (OM). METHODS: Whole exome sequencing was performed on leukemia and non-cancer blood disorder (ncBD) patients' saliva samples (N = 50) prior to conditioning therapy. WHO OM grading scores were determined: moderate to severe (OM2-4) vs. none to mild (OM0-1). Reads were processed using Trim Galorev0.6.7, Bowtie2v2.4.1, Samtoolsv1.10, Genome Analysis Toolkit (GATK)v4.2.6.1, and DeepVariantv1.4.0. We utilized the following pipelines: P1 analysis with PLINK2v3.7, SNP2GENEv1.4.1 and MAGMAv1.07b, and P2 [leukemia (N = 42) vs. ncBDs (N = 8)] and P3 [leukemia + OM2-4 (N = 18) vs. leukemia + OM0-1 (N = 24)] with Z-tests of genotypes and protein-protein interaction determination. GeneCardsSuitev5.14 was used to identify phenotypes (P1 and P2, leukemia; P3, oral mucositis) and average disease-causing likelihood and DGIdb for drug interactions. P1 and P2 genes were analyzed with CytoScape plugin BiNGOv3.0.3 to retrieve overrepresented Gene Ontology (GO) terms and Ensembl's VEP for SNP outcomes. RESULTS: In P1, 457 candidate SNPs (28 genes) were identified and 21,604 SNPs (1016 genes) by MAGMAv1.07b. Eighteen genes were associated with "leukemia" per VarElectv5.14 analysis and predicted to be deleterious. In P2 and P3, 353 and 174 SNPs were significant, respectively. STRINGv12.0 returned 77 and 32 genes (C.L. = 0.7) for P2 and P3, respectively. VarElectv5.14 determined 60 genes from P2 associated with "leukemia" and 11 with "oral mucositis" from P3. Overrepresented GO terms included "cellular process," "signaling," "hemopoiesis," and "regulation of immune response." CONCLUSIONS: We identified candidate SNPs possibly conferring susceptibility to develop leukemia and oral mucositis.

Regulation of STAT1 and STAT4 Expression by Growth Factor and Interferon Supplementation in Sjögren's Syndrome Cell Culture Models.

Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.

Identification of single nucleotide polymorphisms (SNPs) associated with chronic graft-versus-host disease in patients undergoing allogeneic hematopoietic cell transplantation.

INTRODUCTION: Chronic graft-versus-host disease (cGVHD) is a debilitating side effect of allogeneic hematopoietic cell transplantation (HCT), affecting the quality of life of patients. We used whole exome sequencing to identify candidate SNPs and complete a multi-marker gene-level analysis using a cohort of cGVHD( +) (N = 16) and cGVHD( -) (N = 66) HCT patients. METHODS: Saliva samples were collected from HCT patients (N = 82) pre-conditioning in a multi-center study from March 2011 to May 2018. Exome sequencing was performed and FASTQ files were processed for sequence alignments. Significant SNPs were identified by logistic regression using PLINK2v3.7 and Fisher's exact test. One cGVHD( -) patient sample was excluded from further analysis since no SNP was present in at least 10% of the sample population. The FUMA platform's SNP2GENE was utilized to annotate SNPs and generate a MAGMA output. Chromatin state visualization of lead SNPs was completed using Epilogos tool. FUMA's GENE2FUNC was used to obtain gene function and tissue expression from lead genomic loci. RESULTS: Logistic regression classified 986 SNPs associated with cGVHD( +). SNP2GENE returned three genomic risk loci, four lead SNPs, 48 candidate SNPs, seven candidate GWAS tagged SNPs, and four mapped genes. Fisher's exact test identified significant homozygous genotypes of four lead SNPs (p < 0.05). GENE2FUNC analysis of multi-marker SNP sets identified one positional gene set including lead SNPs for KANK1 and KDM4C and two curated gene sets including lead SNPs for PTPRD, KDM4C, and/or KANK1. CONCLUSIONS: Our data suggest that SNPs in three genes located on chromosome 9 confer genetic susceptibility to cGVHD in HCT patients. These genes modulate STAT3 expression and phosphorylation in cancer pathogenesis. The findings may have implications in the modulation of pathways currently targeted by JAK inhibitors in cGVHD clinical trials.

Human oral mucosa and oral microbiome interactions following supragingival plaque reconstitution in healthy volunteers: a diet-controlled balanced design proof-of-concept model to investigate oral pathologies.

Changes in the oral microbiome may contribute to oral pathologies, especially in patients undergoing cancer therapy. Interactions between oral microbiome and oral mucosa may exacerbate inflammation. We determined whether probiotic-controlled plaque formation could impact proximal oral mucosa gene expression profiles in healthy volunteers. A 3-weeks balanced sample collection design from healthy volunteers (HVs) was implemented. At Week-1 plaques samples and labial mucosa brush biopsies were obtained from HVs in the morning (N = 4) and/or in the afternoon (N = 4), and groups were flipped at Week-3. A fruit yogurt and tea diet were given 2-4hrs before sample collection. mRNA gene expression analysis was completed using RNA-Seq and DESeq2. Bacterial taxa relative abundance was determined by 16S HOMINGS. Bacterial diversity changes and metabolic pathway enrichment were determined using PRIMERv7 and LEfSe programs. Alpha- and beta-diversities did not differ morning (AM) vs. afternoon (PM). The most affected KEGG pathway was Toll-like receptor signaling in oral mucosa. Eighteen human genes and nine bacterial genes were differentially expressed in plaque samples. Increased activity for 'caries-free' health-associated calcifying Corynebacterium matruchotii and reduced activity for Aggregatibacter aphrophilus, an opportunistic pathogen, were observed. Microbial diversity was not altered after 8 hours plaque formation in healthy individuals as opposed to gene expression.

Oral microbiomes of patients with infective endocarditis (IE): a comparative pilot study of IE patients, patients at risk for IE and healthy controls.

Background: Infective endocarditis (IE) is an uncommon disease with high morbidity and mortality rates, which often develops from oral bacterial species entering circulation. Objective: We compared oral microbiome profiles of three groups: IE patients (N  9 patients; n = 27 samples), disease controls at risk for IE (N = 28; n = 84), and healthy controls (N = 37; n = 111). Bacterial species in IE patients' blood cultures were identified for comparison with matched oral samples. Design: Oral microbiome profiles were obtained from buccal mucosa, saliva, and tongue samples for all three groups and from sub- and supra-gingival plaque samples of the IE group (N = 9; n = 16) and disease controls (N = 28; n = 54). Alpha- and beta-diversities were determined based on relative abundance data. Discriminative species were identified by LEfSe, post hoc Mann-Whitney, and ROC analyses. Identity of the bacterial species in IE patients' blood cultures was confirmed by 16S-rRNA gene Sanger sequencing. Results: Alpha- and beta-diversities differed between groups. Discriminative IE-associated species were identified, e.g. Haemophilus parainfluenzae and Streptococcus sanguinis. Two blood isolates were Staphylococcus aureus, also identified in one matched saliva sample. Streptococcus mutans was present in one patient's plaque samples and blood culture. Conclusions: Oral microbiomes of IE, non-IE disease controls, and healthy controls differed significantly. A better understanding of IE-related bacterial-host interactions is warranted.

Regulation of MMP9 transcription by ETS1 in immortalized salivary gland epithelial cells of patients with salivary hypofunction and primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) patients exhibit enhanced degradation of the salivary epithelium initially through MMP9 overexpression. We assessed the expression of MMP9 and an associated transcription factor, ETS1, in primary salivary gland epithelial cells (SGECs) and investigated potential regulatory mechanism(s) in immortalized SGECs. SGECs and iSGECs were derived from pSS and/or xerostomic "sicca" patients. siRNA knockdown of ETS1 in iSGECs was performed to determine MMP9 mRNA (qRT-PCR) and protein expression (ELISA). ETS1 binding to MMP9 promoter was assessed by luciferase activity and binding confirmed by mutagenesis and ChIP. Effects of ETS1 overexpression on progenitor and Epithelial-Mesenchymal transition (EMT) associated markers were determined by Western blot. Expression of ETS1 and its phosphorylated form in iSGECs was determined by immunofluorescence microscopy. ETS1 and MMP9 were overexpressed in SGECs of pSS and non-pSS sicca patients with salivary gland lymphocytic infiltration compared to non-pSS sicca patients without infiltration. ETS1 siRNA knockdown reduced both MMP9 mRNA and protein levels. ETS1 overexpression affected the expression of EMT and progenitor cell markers. Lastly, ETS1 bound the MMP9 promoter within the DNA region of -296 bp to -339 bp. ETS1 may impair salivary function through direct transcriptional control of the MMP9 promoter. ETS1 upregulation may also affect other factors involved in repair of the dysfunctional pSS salivary epithelium.

Cutaneous Microbiome Profiles Following Chlorhexidine Treatment in a 72-Hour Daily Follow-Up Paired Design: a Pilot Study.

Venous catheter-related bloodstream infections represent a significant problem in the United States. Our objective was to determine daily changes in skin microbiome profiles up to 72h postchlorhexidine treatment. Left and right forearm skin swab samples were obtained from 10 healthy volunteers over 72h at 24h intervals. Dorsal surface of left arm was treated with chlorohexidine gluconate (CHG) at initial time point (T = 0), while the right arm remained untreated (control). Swab samples were obtained shortly before (T = 0) and after CHG treatment (T = 24-48-72h). Bacterial DNA extraction, 16S rRNA gene V1-V3 sequencing and taxonomic annotation were performed using ZymoBIOMICS pipeline. PERMANOVA, linear discriminant and bacterial interaction network analyses were performed. A total of 13 total phyla, 273 genera, and 950 total species were detected across all time points, CHG-treated or CHG-untreated. Most abundant species included Cutibacterium acnes, Staphylococcus epidermidis, and Rothia Mucilaginosa. Low biomass-related inconsistent taxa detection was observed. PERMANOVA suggested a marginal difference between CHG-treated and CHG-untreated microbiome profiles (Genera: P(perm) = 0.0531; Species: P(perm) = 0.0450). Bacterial interaction network guided PERMANOVA analyses detected a microbiome change over time, suggesting a consistent CHG treatment-specific change. LEfSe identified Finegoldia magna, Bacillus pumilus, Bacillus thermoamylovorans as the only distinctive species. These species were more abundant and/or present post-CHG treatment in the CHG-treated group. These findings suggest that the skin microbiome was not significantly different 24, 48, or 72h after CHG treatment. Previous culture-based studies have found similar results after 24h. Future studies will be needed to determine the mechanisms of bacterial regrowth after CHG treatment. IMPORTANCE Annually, over 80,000 central line infections occur in the United States. Understanding the pathogenesis of these infections is crucial. Chlorhexidine is the most commonly used skin preparation before line placement. We hypothesized that the use of chlorhexidine and dressings will alter the normal arm skin microbiome over a period of 72h. We used 16S-rRNA gene next generation sequencing (NGS) to determine the forearm skin microbiome of volunteers. The left arm was swabbed with chlorhexidine and the right arm served as control. The skin microbiome returned to normal after 24h. Our NGS results confirm findings of two previous culture-based studies. Relative abundance of Bacillus spp. in the chlorhexidine-treated samples was increased, consistent with one previous study. Based on the results of this pilot study, we will need to measure viable bacteria during a 24h time course following chlorhexidine treatment to understand the source of skin microbiome replenishment.

A Computational Text Mining-Guided Meta-Analysis Approach to Identify Potential Xerostomia Drug Targets.

Xerostomia (subjective complaint of dry mouth) is commonly associated with salivary gland hypofunction. Molecular mechanisms associated with xerostomia pathobiology are poorly understood, thus hampering drug development. Our objectives were to (i) use text-mining tools to investigate xerostomia and dry mouth concepts, (ii) identify associated molecular interactions involving genes as candidate drug targets, and (iii) determine how drugs currently used in clinical trials may impact these genes and associated pathways. PubMed and PubMed Central were used to identify search terms associated with xerostomia and/or dry mouth. Search terms were queried in pubmed2ensembl. Protein-protein interaction (PPI) networks were determined using the gene/protein network visualization program search tool for recurring instances of neighboring genes (STRING). A similar program, Cytoscape, was used to determine PPIs of overlapping gene sets. The drug-gene interaction database (DGIdb) and the clinicaltrials.gov database were used to identify potential drug targets from the xerostomia/dry mouth PPI gene set. We identified 64 search terms in common between xerostomia and dry mouth. STRING confirmed PPIs between identified genes (CL = 0.90). Cytoscape analysis determined 58 shared genes, with cytokine-cytokine receptor interaction representing the most significant pathway (p = 1.29 × 10-23) found in the Kyoto encyclopedia of genes and genomes (KEGG). Fifty-four genes in common had drug interactions, per DGIdb analysis. Eighteen drugs, targeting the xerostomia/dry mouth PPI network, have been evaluated for xerostomia, head and neck cancer oral complications, and Sjögren's Syndrome. The PPI network genes IL6R, EGFR, NFKB1, MPO, and TNFSF13B constitute a possible biomarker signature of xerostomia. Validation of the candidate biomarkers is necessary to better stratify patients at the genetic and molecular levels to facilitate drug development or to monitor response to treatment.

Comorbidities and Susceptibility to COVID-19: A Generalized Gene Set Data Mining Approach.

The COVID-19 pandemic has led to over 2.26 million deaths for almost 104 million confirmed cases worldwide, as of 4 February 2021 (WHO). Risk factors include pre-existing conditions such as cancer, cardiovascular disease, diabetes, and obesity. Although several vaccines have been deployed, there are few alternative anti-viral treatments available in the case of reduced or non-existent vaccine protection. Adopting a long-term holistic approach to cope with the COVID-19 pandemic appears critical with the emergence of novel and more infectious SARS-CoV-2 variants. Our objective was to identify comorbidity-associated single nucleotide polymorphisms (SNPs), potentially conferring increased susceptibility to SARS-CoV-2 infection using a computational meta-analysis approach. SNP datasets were downloaded from a publicly available genome-wide association studies (GWAS) catalog for 141 of 258 candidate COVID-19 comorbidities. Gene-level SNP analysis was performed to identify significant pathways by using the program MAGMA. An SNP annotation program was used to analyze MAGMA-identified genes. Differential gene expression was determined for significant genes across 30 general tissue types using the Functional and Annotation Mapping of GWAS online tool GENE2FUNC. COVID-19 comorbidities (n = 22) from six disease categories were found to have significant associated pathways, validated by Q-Q plots (p < 0.05). Protein-protein interactions of significant (p < 0.05) differentially expressed genes were visualized with the STRING program. Gene interaction networks were found to be relevant to SARS and influenza pathogenesis. In conclusion, we were able to identify the pathways potentially affected by or affecting SARS-CoV-2 infection in underlying medical conditions likely to confer susceptibility and/or the severity of COVID-19. Our findings have implications in future COVID-19 experimental research and treatment development.

Haemophilus pittmaniae and Leptotrichia spp. Constitute a Multi-Marker Signature in a Cohort of Human Papillomavirus-Positive Head and Neck Cancer Patients.

OBJECTIVES: Human papillomavirus (HPV) is a known etiological factor of oropharyngeal head and neck cancer (HNC). HPV positivity and periodontal disease have been associated with higher HNC risk, suggesting a role for oral bacterial species. Our objective was to determine oral microbiome profiles in HNC patients (HPV-positive and HPV-negative) and in healthy controls (HC). METHODS: Saliva samples and swabs of buccal mucosa, supragingival plaque, and tongue were collected from HNC patients (N = 23 patients, n = 92 samples) before cancer therapy. Next-generation sequencing (16S-rRNA gene V3-V4 region) was used to determine bacterial taxa relative abundance (RA). ß-Diversities of HNC HPV+ (N = 16 patients, n = 64 samples) and HNC HPV- (N = 7 patients, n = 28 samples) groups were compared using PERMANOVA (pMonte Carlo < 0.05). LEfSe discriminant analysis was performed to identify differentiating taxa (Log LDA > 2.0). RA differences were analyzed by Mann-Whitney U-test (α = 0.05). CombiROC program was used to determine multi-marker bacterial signatures. The Microbial Interaction Network Database (MIND) and LitSuggest online tools were used for complementary analyses. RESULTS: HNC vs. HC and HNC HPV+ vs. HNC HPV- ß-diversities differed significantly (pMonte Carlo < 0.05). Streptococcus was the most abundant genus for HNC and HC groups, while Rothia mucilaginosa and Haemophilus parainfluenzae were the most abundant species in HNC and HC patients, respectively, regardless of antibiotics treatment. LEfSe analysis identified 43 and 44 distinctive species for HNC HPV+ and HNC HPV- groups, respectively. In HNC HPV+ group, 26 periodontal disease-associated species identified by LefSe had a higher average RA compared to HNC HPV- group. The significant species included Alloprevotella tannerae, Fusobacterium periodonticum, Haemophilus pittmaniae, Lachnoanaerobaulum orale, and Leptotrichia spp. (Mann-Whitney U-test, p < 0.05). Of 43 LEfSe-identified species in HPV+ group, 31 had a higher RA compared to HPV- group (Mann-Whitney U-test, p < 0.05). MIND analysis confirmed interactions between Haemophilus and Leptotrichia spp., representing a multi-marker signature per CombiROC analysis [area under the curve (AUC) > 0.9]. LitSuggest correctly classified 15 articles relevant to oral microbiome and HPV status. CONCLUSION: Oral microbiome profiles of HNC HPV+ and HNC HPV- patients differed significantly regarding periodontal-associated species. Our results suggest that oral bacterial species (e.g., Leptotrichia spp.), possessing unique niches and invasive properties, coexist with HPV within HPV-induced oral lesions in HNC patients. Further investigation into host-microbe interactions in HPV-positive HNC patients may shed light into cancer development.

Oral Microbiome Signatures in Hematological Cancers Reveal Predominance of Actinomyces and Rothia Species.

The endogenous microbiome of healthy individuals in oral cavities is diverse, representing over 700 bacterial species. Imbalance in oral and gut microbiome composition and associated gene expression has been linked to different forms of hematological (blood) cancers. Our objective is to compare oral microbiome profiles of patients with blood cancers (BC group: N = 39 patients, n = 124 oral samples) to those of healthy control subjects (HC group: N = 27 subjects, n = 100 oral samples). Saliva samples and swabs of buccal mucosa, supragingival plaque, and tongue were collected from blood cancer patients and healthy controls. Next-generation sequencing (16S-rRNA gene V3-V4 region) was used to determine the relative abundance of bacterial taxa present at the genus and species levels. Differences in oral microbiome beta-diversity were determined using multivariate permutational analysis of variance (PERMANOVA). Linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed to identify differentiating bacterial taxa in pairwise comparisons. The PATRICv3.6.7 online tool was used to determine the predominance of potential pathogenicity in the BC group. The oral microbiome beta-diversities of the BC and HC groups differed and corresponded to a reduced alpha-diversity in the BC group. LEfSe analysis showed significant LDA scores for Actinomyces and Rothia spp., differentiating the BC group from the HC group. In silico analysis using PATRICv3.6.7 demonstrated that the groups of bacteria possessing traits of "antibiotic resistance", "oral pathogen", and "virulence" was enriched in the BC group. Although 56% of the BC patients received antibiotics within two weeks of the oral bacterial sampling, Actinomyces genus remained the top differentiating feature in the BC group regardless of the administration of antibiotics, while Rothia dentocariosa was detected as the top differentiating feature in the BC patients who did not receive antibiotics, but not in those who received antibiotics. Further investigation is needed to better understand the interactions of certain oral species with the host immune system to better characterize clinically relevant associations with hematological cancers.

Immortalization of Salivary Gland Epithelial Cells of Xerostomic Patients: Establishment and Characterization of Novel Cell Lines.

Primary Sjögren's Syndrome (pSS) is an autoimmune disease mainly affecting salivary and lacrimal glands. Previous pSS studies have relied on primary cell culture models or cancer cell lines with limited relevance to the disease. Our objective was to generate and characterize immortalized salivary gland epithelial cells (iSGECs) derived from labial salivary gland (LSG) biopsies of pSS patients (focus score > 1) and non-Sjögren's Syndrome (nSS) xerostomic (i.e., sicca) female patients. To characterize iSGECs (n = 3), mRNA expression of specific epithelial and acinar cell markers was quantified by qRT-PCR. Protein expression of characterization markers was determined by immunocytochemistry and Western blot. Secretion of α-amylase by iSGECs was confirmed through colorimetric activity assay. Spheroid formation and associated alterations in expression markers were determined using matrigel-coated cell culture plates. Consistent mRNA and protein expressions of both epithelial and pro-acinar cell markers were observed in all three iSGEC lines. When cultured on matrigel medium, iSGECs formed spheroids, secreted α-amylase after ß-adrenergic stimulation, and expressed multiple acinar cell markers at late passages. One iSGEC line retained adequate cell morphology without a loss of SV40Lt expression and proliferation potential after over 100 passages. In conclusion, our established iSGEC lines represent a viable model for salivary research due to their passaging capacity and maintenance of pro-acinar cell characteristics.

Lasting Gammaproteobacteria profile changes characterized hematological cancer patients who developed oral mucositis following conditioning therapy.

Background: Oral mucositis (OM) is a common side effect of conditioning therapy implemented before hematopoietic stem cell transplantation (HSCT). The role of oral microbiome in OM is not fully elucidated. Objective: To determine oral microbiome profile changes post-conditioning in HSCT patients who developed moderate OM, or mild to no OM. Design: Patient groups were: Muc0-1 with OM-score = 0-1 (43 paired samples) and Muc2 with WHO OM-score = 2 (36 paired samples). Bacterial DNA was isolated from oral samples (saliva, swabs of buccal mucosa, tongue, and supragingival plaque) at pre-conditioning (T 0 ), post-conditioning mucositis onset (T Muc ), and one-year post-conditioning (T Year ). 16S-rRNA gene next-generation sequencing was used to determine the relative abundance (RA) of >700 oral species. Alpha-diversity, beta-diversity and linear discriminant analyses (LDA) were performed Muc2 versus Muc0-1. Results: Muc2 oral microbiome alpha- and beta-diversity differed between T 0 and T Muc . Muc2 alpha-diversity and Muc0-1 beta-diversity did not differ between T 0 and T Year . T 0 to T Muc LDA scores were significant in Muc2 for Gammaproteobacteria. For Muc2 patients, the average RA decreased for Haemophilus parainfluenza, a species known as mucosal surfaces protector, but increased for Escherichia-Shigella genera. Conclusions: Post-conditioning OM might contribute to long-term oral microbiome changes affecting Gammaproteobacteria, in HSCT patients.

Methodology for the development of a national Dental Practice-Based Research Network survey on dentist's beliefs and behaviors concerning antibiotic prophylaxis.

BACKGROUND: Dentists are high prescribers of antibiotics for both treatment and prevention of infection, although there are few guidelines to aid clinicians. Given the worldwide concerns about unnecessary use of antibiotics, there is a need for a better understanding of dentists' use of these drugs for antibiotic prophylaxis (AP) to prevent distant site infections (i.e., infective endocarditis and prosthetic joint infection). OBJECTIVE: The aim of this study was to develop and implement an effective, self-reporting, cross-sectional, survey instrument that optimized the response rate and maximized reliability and validity for determining the beliefs and behaviors of a large and nationally representative group of generalist and specialist dentists concerning their use of AP. STUDY DESIGN: A 15-question survey (58 items) was developed in a structured process by a multidisciplinary team and configured for automated online dissemination to 3584 national Dental Practice-Based Research Network (DPBRN; hitherto referred to as "Network") practitioners. The implementation phase consisted of 3 waves of greater than 1000 Network members. Additionally, 47 randomly selected dentists were surveyed twice to assess test-retest reliability. RESULTS: Of 3584 eligible Network members, 2169 (60.5%) completed the survey. The age and geographic distributions of responders was similar to those of dentists in the 2019 American Dental Association census. Furthermore, test-retest weighted kappa values for the survey were acceptable (median 0.56; interquartile range 0.42-0.64). CONCLUSIONS: We developed a highly structured survey with a high response rate and good reliability that will allow us to obtain unique data on dentists' beliefs and practices regarding AP prescribing.

Identification of single nucleotide pleomorphisms associated with periodontal disease in head and neck cancer irradiation patients by exome sequencing.

OBJECTIVE: Periodontal disease (PD) is a common oral complication in patients with head and neck cancer (HNC) undergoing radiation therapy (RT). Our objective was to identify candidate single nucleotide polymorphisms (SNPs) associated with PD in radiation-treated patients with HNC. STUDY DESIGN: DNA was extracted from the saliva of patients with HNC (n = 69) before RT. Clinical attachment loss (CAL) increment greater than 0.2 mm over 24 months after RT was used to define PD progression. After exome sequencing, SNPs associated with post-RT PD progression were identified by using logistic regression and homozygosity analyses. The web tools STRING, the Database for Annotation, Visualization and Integrated Discovery (DAVID), GeneCodis, and Ensembl Variant Effect Predictor were used for functional analysis. RESULTS: Of the 48 patients with HNC with post-RT PD progression, 24 had no tooth with 5 mm or greater pocket depth before RT, whereas of the 21 patients with HNC without progression, 11 had PD initially. A total of 330 SNPs (249 genes) with over-represented homozygous genotype (98.5% variant allele) were found to be associated with post-RT PD. Sixty of these corresponded to PD-related pathways, including previously identified genes. In patients with HNC with post-RT PD progression, SNPs were found in genes (n = 10) in contrast to those without progression (n = 7). CONCLUSIONS: The SNPs of collagen genes were identified, potentially defining susceptibility to PD in patients with HNC, and this could be further investigated to characterize PD drug targets.

Telomere erosion in Sjögren's syndrome: A multi-tissue comparative analysis.

BACKGROUND: Acinar progenitor cells within salivary glands have decreased regenerative capacity and exhibit shorter telomeres in primary Sjögren's syndrome (pSS) patients. We investigated whether DNA of saliva, PBMCs, and labial salivary gland (LSG) biopsy tissue have shorter telomeres in pSS compared to controls. mRNA expression of genes associated with pSS pathogenesis (ETS1, LEF1, and MMP9), telomere DNA damage response (ATM), senescence (CDKN2A), telomerase inhibition (IFN-y, TGFß1), and the shelterin complex (TPP1, POT1) were assessed in LSG tissue by qRT-PCR to examine potential defects in telomere maintenance. METHODS: Relative telomere length in DNA of saliva, PBMCs, and LSGs from non-pSS sicca and pSS patients was measured using qPCR. Saliva DNA telomere length was further compared to healthy controls. Expression of genes affecting telomere maintenance was analyzed in LSGs using qRT-PCR. RESULTS: Primary Sjögren's syndrome patients have shorter telomeres in saliva DNA (n = 21) than healthy controls (n = 27) (P = .0035). ATM mRNA expression was higher in pSS LSG tissue (n = 16) vs non-pSS sicca patients (n = 13) (P = .0283) and strongly correlated with LEF1, TPP1, and POT1 (P < .01, r > 0.6). CONCLUSIONS: Patients with pSS exhibited significant telomere erosion in saliva DNA. Overexpression of ATM in LSGs could represent a compensatory response to telomere shortening. The role of LEF1 in telomere erosion remains to be elucidated.

Oral Microbiome and Cancer Therapy-Induced Oral Mucositis.

Characterization of the role of oral microbiome in cancer therapy-induced oral mucositis (CTOM) is critical in preventing the clinically deleterious effects on patients' health that are associated with CTOM. Funding initiatives related to the National Institutes of Health human microbiome project have resulted in groundbreaking advancements in biology and medicine during the last decade. These advancements have shown that a human being is in fact a superorganism made of human cells and associated symbiotic or commensal microbiota. In this review, we describe the state of science as it relates to fundamental knowledge on oral microbiome and its role in CTOM. We also discuss how state-of-the-art technologies and systems biology tools may be used to help tackle the difficult challenges ahead to develop effective treatments or preventive therapies for oral mucositis. We make a clear distinction between disease processes pertaining to the oral microbiome, which includes opportunistic pathogens that may be defined as pathobionts, and those infectious disease processes initiated by exogenous pathogens. We also explored the extent to which knowledge from the gastrointestinal tract in disease and intestinal mucositis could help us better understand CTOM pathobiology. Finally, we propose a model in which the oral microbiome participates in the current five-step CTOM pathobiology model. With the advent of more sophisticated metagenomics technologies and methods of analysis, much hope lies ahead to implement an effective holistic approach to treat cancer patients affected by CTOM.

Caries-associated oral microbiome in head and neck cancer radiation patients: a longitudinal study.

Head and neck cancer (HNC) therapy often leads to caries development. Our goal was to characterize the oral microbiome of HNC patients who underwent radiation therapy (RT) at baseline (T0), and 6 (T6) and 18 (T18) months post-RT, and to determine if there was a relationship with increased caries. HOMINGS was used to determine the relative abundance (RA) of >600 bacterial species in oral samples of 31 HNC patients. The DMFS score was used to define patient groups with tooth decay increase (DMFS[+]) or no increase (DMFS[-]).A change in microbiome beta-diversity was observed at T6 and T18. The Streptococcus mutans RA increased at T6 in both DMFS[+] and DMFS[-] groups. The RA of Prevotella melaninogenica, the species often associated with caries in young children, decreased at T6 in the DMFS[-] group. The RA of the health-associated species, Abiotrophia defective, decreased in the DMFS[+] group. The oral microbiome underwent significant changes in radiation-treated HNC patients, whether they developed caries or not. Caries rates were not associated with a difference in salivary flow reduction between DMFS[+] andDMFS[-] groups. Patients who develop caries might be more susceptible to certain species associated with oral disease or have fewer potentially protective oral species.

Expression of ETS1 and LEF1 in salivary glands of Sjögren syndrome patients.

OBJECTIVE: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease affecting exocrine glands, thereby causing dry mouth and eyes (sicca). Our objective was to determine the expression of pSS pathogenic biomarker MMP9 and its putative transcription factors ETS1 and LEF1, in labial salivary glands of pSS patients. METHODS: Sicca patients were assigned to three groups based on focus score (FS): non-pSS sicca (i.e., GR1 [FS = 0] and GR2 [0 < FS < 1]) and pSS (i.e., GR3 [FS ≥ 1]). We determined the mRNA and protein expression of MMP9, ETS1, and LEF1 in salivary gland biopsies. Also, ETS1-CD4 and LEF1-CD4 co-expression analyses were performed. RESULTS: The mRNA expression of MMP9, ETS1, and LEF1 was upregulated in GR3 compared to GR1 (p < 0.01). Most GR3 salivary gland areas had moderate to high MMP9, ETS1, and LEF1 protein expression compared to GR1 and GR2. Further, ETS1-CD4 and LEF1-CD4 dual staining demonstrated that both salivary gland epithelial cells and lymphocytic infiltrates had increased levels of ETS1 and LEF1. Moreover, there was a strong correlation between ETS1(+)-CD4(-) and LEF1(+)-CD4(-) cells. CONCLUSION: These results suggest, for the first time, a concerted increase in ETS1 and LEF1 expression in salivary gland epithelial cells of pSS patients that is reflective of the etiopathogenesis of pSS.

Text mining-based in silico drug discovery in oral mucositis caused by high-dose cancer therapy.

INTRODUCTION: Oral mucositis (OM) is a major dose-limiting side effect of chemotherapy and radiation used in cancer treatment. Due to the complex nature of OM, currently available drug-based treatments are of limited efficacy. OBJECTIVES: Our objectives were (i) to determine genes and molecular pathways associated with OM and wound healing using computational tools and publicly available data and (ii) to identify drugs formulated for topical use targeting the relevant OM molecular pathways. METHODS: OM and wound healing-associated genes were determined by text mining, and the intersection of the two gene sets was selected for gene ontology analysis using the GeneCodis program. Protein interaction network analysis was performed using STRING-db. Enriched gene sets belonging to the identified pathways were queried against the Drug-Gene Interaction database to find drug candidates for topical use in OM. RESULTS: Our analysis identified 447 genes common to both the "OM" and "wound healing" text mining concepts. Gene enrichment analysis yielded 20 genes representing six pathways and targetable by a total of 32 drugs which could possibly be formulated for topical application. A manual search on ClinicalTrials.gov confirmed no relevant pathway/drug candidate had been overlooked. Twenty-five of the 32 drugs can directly affect the PTGS2 (COX-2) pathway, the pathway that has been targeted in previous clinical trials with limited success. CONCLUSIONS: Drug discovery using in silico text mining and pathway analysis tools can facilitate the identification of existing drugs that have the potential of topical administration to improve OM treatment.