Induction of Endogenous Antimicrobial Peptides to Prevent or Treat Oral Infection and Inflammation.

Antibiotics are often used to treat oral infections. Unfortunately, excessive antibiotic use can adversely alter oral microbiomes and promote the development of antibiotic-resistant microorganisms, which can be difficult to treat. An alternate approach could be to induce the local transcription and expression of endogenous oral antimicrobial peptides (AMPs). To assess the feasibility and benefits of this approach, we conducted literature searches to identify (i) the AMPs expressed in the oral cavity; (ii) the methods used to induce endogenous AMP expression; and (iii) the roles that expressed AMPs may have in regulating oral inflammation, immunity, healing, and pain. Search results identified human neutrophil peptides (HNP), human beta defensins (HBD), and cathelicidin AMP (CAMP) gene product LL-37 as prominent AMPs expressed by oral cells and tissues. HNP, HBD, and LL-37 expression can be induced by micronutrients (trace elements, elements, and vitamins), nutrients, macronutrients (mono-, di-, and polysaccharides, amino acids, pyropeptides, proteins, and fatty acids), proinflammatory agonists, thyroid hormones, and exposure to ultraviolet (UV) irradiation, red light, or near infrared radiation (NIR). Localized AMP expression can help reduce infection, inflammation, and pain and help oral tissues heal. The use of a specific inducer depends upon the overall objective. Inducing the expression of AMPs through beneficial foods would be suitable for long-term health protection. Additionally, the specialized metabolites or concentrated extracts that are utilized as dosage forms would maintain the oral and intestinal microbiome composition and control oral and intestinal infections. Inducing AMP expression using irradiation methodologies would be applicable to a specific oral treatment area in addition to controlling local infections while regulating inflammatory and healing processes.

Antimicrobial Peptides and Biomarkers Induced by Ultraviolet Irradiation Have the Potential to Reduce Endodontic Inflammation and Facilitate Tissue Healing.

BACKGROUND: Ultraviolet (UV) irradiation can modulate host immune responses and this approach is a novel application for treating endodontic infections and inflammation in root canals. METHODS: A dataset of UV-induced molecules was compiled from a literature search. A subset of this dataset was used to calculate expression log2 ratios of endodontic tissue molecules from HEPM cells and gingival fibroblasts after 255, 405, and 255/405 nm UV irradiation. Both datasets were analyzed using ingenuity pathway analysis (IPA, Qiagen, Germantown, MD, USA). Statistical significance was calculated using Fisher's exact test and z-scores were calculated for IPA comparison analysis. RESULTS: The dataset of 32 UV-induced molecules contained 9 antimicrobial peptides, 10 cytokines, 6 growth factors, 3 enzymes, 2 transmembrane receptors, and 2 transcription regulators. These molecules were in the IPA canonical pathway annotations for the wound healing signaling pathway (9/32, p = 3.22 × 10-11) and communication between immune cells (6/32, p = 8.74 × 10-11). In the IPA disease and function annotations, the 32 molecules were associated with an antimicrobial response, cell-to-cell signaling and interaction, cellular movement, hematological system development and function, immune cell trafficking, and inflammatory response. In IPA comparison analysis of the 13 molecules, the predicted activation or inhibition of pathways depended upon the cell type exposed, the wavelength of the UV irradiation used, and the time after exposure. CONCLUSIONS: UV irradiation activates and inhibits cellular pathways and immune functions. These results suggested that UV irradiation can activate innate and adaptive immune responses, which may supplement endodontic procedures to reduce infection, inflammation, and pain and assist tissues to heal.

Dataset of endodontic microorganisms killed at 265 nm wavelength by an ultraviolet C light emitting diode in root canals of extracted, instrumented teeth.

Ultraviolet C (UVC) light emitting diode (LED) can kill the endodontic pathogen Enterococcus faecalis and has the potential to kill other oral microorganisms associated with endodontic infections. This same bacteriocidal device shows great promise in the stimulation of periapical healing and pain reduction resulting from inflammation in root canals. Previously, we found that 255 nm UVC LED killed E. faecalis and induced the production of cellular biomarkers in HEPM cells and gingival fibroblasts (Morio et al., 2019). Here, we extend those findings and hypothesize that UVC LED at other wavelengths and power levels kill microorganisms associated with root canal infections. Units emitting UVC LED at 265 nm (12 mW), 265 nm (22.5 mW), and 280 nm (8 mW) wavelenths were assembled and the energy levels of their emissions were measured. The energy doses in millijoules (mJ) were calculated from the power readings of the meter (µW) × time of exposure (seconds). Ex vivo models of root canals were prepared in extracted, instrumented, single canal human premolars. Five cultures of microorganisms were treated with 265 nm (12 mW), 265 nm (22.5 mW), or 280 nm (8 mW) UVC LED on discs in laboratory assays and 4 cultures of microorganisms were treated with 265 nm (22.5 mW) UVC LED in root canals of extracted, instrumented teeth. After UVC LED treatment, all microorganisms were cultivated on microbiological media. Colony forming units (CFU) of viable microorganisms treated with UVC LED were counted and compared with those of viable microorganisms not treated with UVC LED as controls. Tukey's Honestly Significant Difference was used to determine statistical significances (0.05). Units emitting UVC LED at 265 nm (12 mW), 265 nm (22.5 mW), and 280 nm (8 mW) killed Candida albicans, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), E. faecalis, and Streptococcus sanguinis after 30-90 seconds of exposure in laboratory assays (p < 0.05). Microbial killing differed among treatment times, UVC LED wavelengths, power levels of each unit, and specific microorganism. The unit emitting UVC LED at 265 nm (22.5 mW) killed C. albicans, S. aureus, MRSA, and E. faecalis in 30 s in root canals of extracted, instrumented teeth (p < 0.05). This dataset can be reused to assess the ability of other wavelengths and power levels to kill microorganisms as well as improve procedures for treating endodontic infections and inflammation in root canals.

Dataset-chemokines, cytokines, and biomarkers in the saliva of children with Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease that can also occur in children. The disease is not well defined and there is limited information on the presence of chemokines, cytokines, and biomarkers (CCBMs) in the saliva of children that could improve their disease diagnosis. In a recent study [1], we reported a large dataset of 105 CCBMs that were associated with both lymphocyte and mononuclear cell functions [2] in the saliva of 11 children formally diagnosed with Sjögren's syndrome and 16 normal healthy children. Here, we extend those findings and use the Mendeley dataset [2] to identify CCBMs that have predictive power for Sjögren's syndrome in female children. Datasets of CCBMs from all saliva samples and female children saliva samples were standardized. We used machine learning methods to select Sjögren's syndrome associated CCBMs and assessed the predictive power of selected CCBMs in these two datasets using receiver operating characteristic (ROC) curves and associated areas under curve (AUC) as metrics. We used eight classifiers to identify 16 datasets that contained from 2 to 34 CCBMs with AUC values ranging from 0.91 to 0.94.

A distinguishing profile of chemokines, cytokines and biomarkers in the saliva of children with Sjögren's syndrome.

OBJECTIVE: SS is an autoimmune disease most commonly diagnosed in adults but can occur in children. Our objective was to assess the presence of chemokines, cytokines and biomarkers (CCBMs) in saliva from these children that were associated with lymphocyte and mononuclear cell functions. METHODS: Saliva was collected from 11 children diagnosed with SS prior to age 18 years and 16 normal healthy children. A total of 105 CCBMs were detected in multiplex microparticle-based immunoassays. ANOVA and t test (0.05 level) were used to detect differences. Ingenuity Pathway Analysis (IPA) was used to assess whether elevated CCBMs were in annotations associated with immune system diseases and select leukocyte activities and functions. Machine learning methods were used to evaluate the predictive power of these CCBMs for SS and were measured by receiver operating characteristic (ROC) curve and area under curve (AUC). RESULTS: Of the 105 CCBMs detected, 43 (40.9%) differed in children with SS from those in healthy study controls (P < 0.05) and could differentiate the two groups (P < 0.05). Elevated CCBMs in IPA annotations were associated with autoimmune diseases and with leukocyte chemotaxis, migration, proliferation, and regulation of T cell activation. The best AUC value in ROC analysis was 0.93, indicating that there are small numbers of CCBMs that may be useful for diagnosis of SS. CONCLUSION: While 35 of these 43 CCBMs have been previously reported in SS, 8 CCBMs had not. Additional studies focusing on these CCBMs may provide further insight into disease pathogenesis and may contribute to diagnosis of SS in children.

PD-L1 correlates with chemokines and cytokines in gingival crevicular fluid from healthy and diseased sites in subjects with periodontitis.

OBJECTIVE: PD-L1 is an immune checkpoint molecule that regulates immune and inflammatory responses. While cells of periodontal tissues express PD-L1, its presence in GCF is not known. The purpose of this study was to measure the PD-L1 values in GCF and correlate values with the presence of chemokine and cytokine values from periodontally diseased subjects and periodontally healthy subjects. RESULTS: PD-L1 values (pg/30 s), determined in triplicate using a fluorescent microparticle-based immunoassay ranged from 0.04-31.65 pg/30 s. PD-L1 correlated with 15 out of 22 chemokine and cytokine responses. In 85 healthy sites in 31 subjects, PD-L1 values were negatively correlated with IL6, CXCL8, IL10, and CCL3 values. In 53 diseased sites in 20 subjects, PD-L1 values were positively correlated with CCL11, CSF2, IFNG, IL1A, IL1B, IL2, IL7, IL15, and CCL5 values and negatively correlated with IL12A and IL5 values. Gene ontology (GO) annotations identified roles of PD-L1 in Th1 and Th2 activation and T-cell exhaustion signaling canonical pathways. PD-L1 values were correlated with the expression of chemokines and cytokines, which likely regulates immune cell trafficking and protects the periodontium from uncontrolled immune responses to pathogens and inflammation-induced tissue damage.

Antimicrobial Prosthetic Surfaces in the Oral Cavity-A Perspective on Creative Approaches.

Replacement of missing teeth is an essential component of comprehensive dental care for patients suffering of edentulism. A popular option is implant-supported restorations. However, implant surfaces can become colonized with polymicrobial biofilms containing Candida species that may compromise peri-implant health. To prevent this, implant components may be treated with a variety of coatings to create surfaces that either repel the attachment of viable microorganisms or kill microorganisms on contact. These coatings may consist of nanoparticles of pure elements (more commonly silver, copper, and zinc), sanitizing agents and disinfectants (quaternary ammonium ions and chlorhexidine), antibiotics (cefalotin, vancomycin, and gentamicin), or antimicrobial peptides (AMPs). AMPs in bioactive coatings have a number of advantages. They elicit a protective action against pathogens, inhibit the formation of biofilms, are less toxic to host tissues, and do not prompt inflammatory responses. Furthermore, many of these coatings may involve unique delivery systems to direct their antimicrobial capacity against pathogens, but not commensals. Coatings may also contain multiple antimicrobial substances to widen antimicrobial activity across multiple microbial species. Here, we compiled relevant information about a variety of creative approaches used to generate antimicrobial prosthetic surfaces in the oral cavity with the purpose of facilitating implant integration and peri-implant tissue health.

HBD3 Induces PD-L1 Expression on Head and Neck Squamous Cell Carcinoma Cell Lines.

Human ß-defensin 3 (HBD3) is an antimicrobial peptide up-regulated in the oral tissues of individuals with head and neck squamous cell carcinomas (HNSCC) and oral squamous cell carcinomas (SCC) and present in high concentrations in their saliva. In this study, we determined if HBD3 contributes to HNSCC pathogenesis by inducing programmed death-ligand 1 (PD-L1) expression on HNSCC cell lines. For this, SCC cell lines SCC4, SCC15, SCC19, SCC25, and SCC99 (5.0 × 104 viable cells) were used. Cells were incubated with IFNγ (0.6 µM) and HBD3 (0.2, 2.0, or 20.0 µM) for 24 h. Cells alone served as controls. Cells were then treated with anti-human APC-CD274 (PD-L1) and Live/Dead Fixable Green Dead Cell Stain. Cells treated with an isotype antibody and cells alone served as controls. All cell suspensions were analyzed in a LSR II Violet Flow Cytometer. Cytometric data was analyzed using FlowJo software. Treatment with IFNγ (0.6 µM) increased the number of cells expressing PD-L1 (p < 0.05) with respect to controls. Treatment with HBD3 (20.0 µM) also increased the number of cells expressing PD-L1 (p < 0.05) with respect to controls. However, treatment with IFNγ (0.6 µM) was not significantly different from treatment with HBD3 (20.0 µM) and the numbers of cells expressing PD-L1 were similar (p = 1). Thus, HBD3 increases the number of cells expressing PD-L1. This is a novel concept, but the role HBD3 contributes to HNSCC pathogenesis by inducing PD-L1 expression in tumors will have to be determined.

Computational Models Accurately Predict Multi-Cell Biomarker Profiles in Inflammation and Cancer.

Individual computational models of single myeloid, lymphoid, epithelial, and cancer cells were created and combined into multi-cell computational models and used to predict the collective chemokine, cytokine, and cellular biomarker profiles often seen in inflamed or cancerous tissues. Predicted chemokine and cytokine output profiles from multi-cell computational models of gingival epithelial keratinocytes (GE KER), dendritic cells (DC), and helper T lymphocytes (HTL) exposed to lipopolysaccharide (LPS) or synthetic triacylated lipopeptide (Pam3CSK4) as well as multi-cell computational models of multiple myeloma (MM) and DC were validated using the observed chemokine and cytokine responses from the same cell type combinations grown in laboratory multi-cell cultures with accuracy. Predicted and observed chemokine and cytokine responses of GE KER + DC + HTL exposed to LPS and Pam3CSK4 matched 75% (15/20, p = 0.02069) and 80% (16/20, P = 0.005909), respectively. Multi-cell computational models became 'personalized' when cell line-specific genomic data were included into simulations, again validated with the same cell lines grown in laboratory multi-cell cultures. Here, predicted and observed chemokine and cytokine responses of MM cells lines MM.1S and U266B1 matched 75% (3/4) and MM.1S and U266B1 inhibition of DC marker expression in co-culture matched 100% (6/6). Multi-cell computational models have the potential to identify approaches altering the predicted disease-associated output profiles, particularly as high throughput screening tools for anti-inflammatory or immuno-oncology treatments of inflamed multi-cellular tissues and the tumor microenvironment.

255-nm Light-emitting Diode Kills Enterococcus faecalis and Induces the Production of Cellular Biomarkers in Human Embryonic Palatal Mesenchyme Cells and Gingival Fibroblasts.

INTRODUCTION: The successful treatment of infected or inflamed endodontic tissues requires chemomechanical debridement of the canal spaces and proper sealing of the coronal and apical canal openings. Only a few methods are available to further disinfect areas or initiate regeneration of local tissues. In this study, we assessed the ability of 255-nm and 405-nm light-emitting diode (LED) treatment to kill planktonic cultures of Enterococcus faecalis and induce the production of cellular biomarkers related to endodontic tissue regeneration. METHODS: We determined the antimicrobial effects of 255-nm and 405-nm LED treatment on E. faecalis and the effects of 255-nm and 405-nm LED treatment on the production of osteoinductive, angiogenic, proliferative, and proinflammatory biomarkers from human embryonic palatal mesenchyme (HEPM) cells and gingival fibroblasts. RESULTS: We showed that 255-nm LED but not 405-nm LED treatment killed E. faecalis; the 255-nm LED and sodium hypochlorite more efficiently killed E. faecalis; neither 255-nm nor 405-nm LED treatment affected the viability of HEPM cells and gingival fibroblasts; and 255-nm LED treatment, alone or in combination with 405-nm LED treatment, of HEPM cells and gingival fibroblasts induced the production of biomarkers related to endodontic tissue regeneration. CONCLUSIONS: The results of this study suggest a new treatment modality using short periods of 255-nm LED treatment as an adjunct to chemomechanical debridement for the disinfection of inflamed sites and the production of biomarkers related to endodontic tissue regeneration.

Dataset on the chemokine and cytokine responses of multi-cell cultures treated with Porphyromonas gingivalis hemagglutinin B.

Chemokines and cytokines produced in gingival tissues exposed to microorganisms and microbial products in dental plaque lead to local inflammation and tissue damage seen in periodontal disease. Bates et al. 2018 [1] reported that Porphyromonas gingivalis hemagglutinin B (HagB)-induced matrix metalloproteinase (MMP) responses of single cell cultures containing dendritic cells, gingival epithelial (GE) keratinocytes, or T cells were significantly different from the MMP responses of these same cells grown in multi-cell cultures. Here we report the concentrations (pg/ml) of HagB-induced IL1α, IL6, IL8, IL12(p40), GM-CSF, MIP1α, MIP1ß, RANTES, TNFα, and VEGF produced by dendritic cells, GE keratinocytes, or T cells in single cell cultures, two-cell cultures, or three-cell cultures.

Matrix Metalloproteinase Response of Dendritic Cell, Gingival Epithelial Keratinocyte, and T-Cell Transwell Co-Cultures Treated with Porphyromonas gingivalis Hemagglutinin-B.

Matrix metalloproteinases (MMPs) are enzymes involved in periodontal tissue destruction. Hemagglutinin B (HagB) from the periodontal pathogen Porphyromonas gingivalis induces an elevated MMP response in dendritic cells, but responses from cultures of single-cell types do not reflect the local tissue environment. The objective of this study was to measure HagB-induced MMP responses in a transwell co-culture system containing dendritic cells, gingival epithelial (GE) keratinocytes, and CD4+ T-cells. Transwell co-cultures were assembled and treated with or without HagB. Immunoassays were used to determine production of MMP1, MMP7, MMP9, and MMP12 in response to HagB up to 64 h. Control responses were subtracted from HagB-induced responses. A two-way fixed effect ANOVA was fit to log-transformed concentrations and pairwise group comparisons were conducted (p < 0.05). At 64 h, dendritic cells produced elevated MMP1 and MMP9 responses, which were attenuated in the 3-cell co-culture (p < 0.05). There were also significant differences in MMP7 and MMP12 production between single-cell cultures and co-cultures. These results support the need to use multiple cell types in culture models to evaluate a more representative response to proinflammatory agonists. This three-cell transwell co-culture model may help us better understand the inflammatory process in periodontal disease and test novel therapeutic approaches.

Matrix metalloproteinase (MMP) and immunosuppressive biomarker profiles of seven head and neck squamous cell carcinoma (HNSCC) cell lines.

BACKGROUND: Biomarkers like programmed death ligand-1 (PDL1) have become a focal point for immunotherapeutic checkpoint inhibition in head and neck squamous cell carcinoma (HNSCC). However, it's only part of the total immunosuppressive biomarker profile of HNSCC cells. Matrix metalloproteinases (MMPs) are enzymes that break down the basement membrane allowing cancer cells to metastasize and play an important role in the tumor microenvironment. MMPs can also activate certain cytokines, growth factors, and chemokines post-translationally. The objective of this study was to determine MMP and biomarker profiles of seven different HNSCC cell lines. METHODS: Authenticated cell lines were grown in minimal media at 1×106 viable cells/mL and incubated at 37 °C. After 24 hrs supernatants were collected, and adhering cells were lysed. Multiplex immunoassays were used to determine MMP1, MMP7, MMP9, IL-6, VEGFA, IL-1α, TNF-α, GM-CSF, IL-1RA, and IL-8 concentrations in supernatants. ELISAs were used to determine PDL1, CD47, FASL, and IDO concentrations in cell lysates. A one-way ANOVA was fit to examine log-transformed concentrations of biomarkers between seven HNSCC cell lines, and pairwise group comparisons were conducted using post- hoc Tukey's honest significance test (α=0.05). RESULTS: Significant differences (P<0.05) in MMP and biomarker concentrations were found between the seven HNSCC cell lines. For example, MMP9 was highest in SCC25 and UM-SCC99, MMP7 was highest in SCC25 and UM-SCC19, and MMP1 was highest in SCC25. CONCLUSIONS: These results suggest different patients' HNSCC cells can express distinct profiles of select biomarkers and MMPs, which could be due to metastatic stage of the cancer, primary tumor site, type of tissue the tumor originated from, or genomic differences between patients. MMP and biomarker expression profiles should be considered when choosing cell lines for future studies. The results support the reason for personalized medicine and the need to further investigate how it can be used to treat HNSCC.

Correction to: Genomics of NSCLC patients both affirm PD-L1 expression and predict their clinical responses to anti-PD-1 immunotherapy.

It has been highlighted that in the original manuscript [1] Table S3 'An example of the predictive computational modeling process. Specific details on an annexure section of the PD-L1 pathway show the step-by-step reactions, mechanisms, and reaction equations that occur. Such reactions also occurred in all of the other pathways' was omitted and did not appear in the Additional files and that the Additional files were miss-numbered thereafter. This Correction shows the correct and incorrect Additional files. The original article has been updated.

Human beta defensin 3 alters matrix metalloproteinase production in human dendritic cells exposed to Porphyromonas gingivalis hemagglutinin B.

BACKGROUND: Matrix metalloproteinases (MMPs) are zinc- or calcium-dependent proteinases involved in normal maintenance of extracellular matrix. When elevated, they contribute to the tissue destruction seen in periodontal disease. Recently, we found that human beta defensin 3 (HBD3), a cationic antimicrobial peptide, alters chemokine and proinflammatory cytokine responses in human myeloid dendritic cells exposed to Porphyromonas gingivalis hemagglutinin B (HagB). In this study, the hypotheses that HagB induces MMP production in dendritic cells and that HBD3 mixed with HagB prior to treatment alters HagB-induced MMP profiles were tested. METHODS: Dendritic cells were exposed to 0.2 µM HagB alone and HagB + HBD3 (0.2 or 2.0 µM) mixtures. After 16 hours, concentrations of MMPs in cell culture media were determined with commercial multiplex fluorescent bead-based immunoassays. An integrated cell network was used to identify potential HagB-induced signaling pathways in dendritic cells leading to the production of MMPs. RESULTS: 0.2 µM HagB induced MMP1, -2, -7, -9, and -12 responses in dendritic cells. 0.2 µM HBD3 enhanced the HagB-induced MMP7 response (P < 0.05) and 2.0 µM HBD3 attenuated HagB-induced MMP1, -7, and -9 responses (P < 0.05). The MMP12 response was not affected. In the predicted network, MMPs are produced via activation of multiple pathways. Signals converge to activate numerous transcription factors, which transcribe different MMPs. CONCLUSION: HagB was an MMP stimulus and HBD3 was found to decrease HagB-induced MMP1, -7, and -9 responses in dendritic cells at 16 hours, an observation that suggests HBD3 can alter microbial antigen-induced production of MMPs.

Genomics of NSCLC patients both affirm PD-L1 expression and predict their clinical responses to anti-PD-1 immunotherapy.

BACKGROUND: Programmed Death Ligand 1 (PD-L1) is a co-stimulatory and immune checkpoint protein. PD-L1 expression in non-small cell lung cancers (NSCLC) is a hallmark of adaptive resistance and its expression is often used to predict the outcome of Programmed Death 1 (PD-1) and PD-L1 immunotherapy treatments. However, clinical benefits do not occur in all patients and new approaches are needed to assist in selecting patients for PD-1 or PD-L1 immunotherapies. Here, we hypothesized that patient tumor cell genomics influenced cell signaling and expression of PD-L1, chemokines, and immunosuppressive molecules and these profiles could be used to predict patient clinical responses. METHODS: We used a recent dataset from NSCLC patients treated with pembrolizumab. Deleterious gene mutational profiles in patient exomes were identified and annotated into a cancer network to create NSCLC patient-specific predictive computational simulation models. Validation checks were performed on the cancer network, simulation model predictions, and PD-1 match rates between patient-specific predicted and clinical responses. RESULTS: Expression profiles of these 24 chemokines and immunosuppressive molecules were used to identify patients who would or would not respond to PD-1 immunotherapy. PD-L1 expression alone was not sufficient to predict which patients would or would not respond to PD-1 immunotherapy. Adding chemokine and immunosuppressive molecule expression profiles allowed patient models to achieve a greater than 85.0% predictive correlation among predicted and reported patient clinical responses. CONCLUSIONS: Our results suggested that chemokine and immunosuppressive molecule expression profiles can be used to accurately predict clinical responses thus differentiating among patients who would and would not benefit from PD-1 or PD-L1 immunotherapies.

Promise of Combining Antifungal Agents in Denture Adhesives to Fight Candida Species Infections.

PURPOSE: Several complications may arise in patients wearing complete prosthetic appliances, including denture-associated infections and mucosal stomatitis due to Candida species. This study evaluated the activity of anti-Candida agents in denture adhesive and the cytotoxicities of these preparations for primary human gingival epithelial (GE) keratinocytes. MATERIALS AND METHODS: The anti-Candida activities of antimicrobial peptides, antimicrobial lipids, and antifungal agents against C. albicans ATCC 64124 or HMV4C were assessed in microdilution assays containing water or 1% denture adhesive. The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) were determined. The cytotoxicities of denture adhesive compounded with these agents were assessed in 1.0 × 105 primary GE keratinocytes in LGM-3 media with resazurin. RESULTS: Lactoferricin B, SMAP28, sphingosine, dihydrosphingosine, and phytosphingosine in 1% denture adhesive lost antimicrobial activity for C. albicans (p < 0.05). Amphotericin B, chlorhexidine dihydrochloride, chlorhexidine gluconate, fluconazole, and nystatin in 1% denture adhesive or compounded directly into denture adhesive and then diluted to 1% adhesive, did not lose antimicrobial activity. Compounded formulations were not cytotoxic (LD50 > 100.0 µg/ml) against primary human GE keratinocytes. CONCLUSIONS: Antimicrobial peptides and antimicrobial lipids had diminished activities in 1% adhesive, suggesting that components in adhesives may inactivate local innate immune factors in the oral cavity, possibly predisposing denture wearers to Candida species infections. More importantly, antifungal agents retained their anti-C. albicans activities in denture adhesive, strongly suggesting that antifungal agents could be candidates for inclusion in adhesive formulations and used as prescribed topical treatments for individuals with denture stomatitis.

Cell genomics and immunosuppressive biomarker expression influence PD-L1 immunotherapy treatment responses in HNSCC-a computational study.

OBJECTIVES: Programmed death-ligand 1 (PD-L1) expression is correlated with objective response rates to PD-1 and PD-L1 immunotherapies. However, both immunotherapies have only demonstrated 12%-24.8% objective response rates in patients with head and neck squamous cell carcinoma (HNSCC), demonstrating a need for a more accurate method to identify those who will respond before their therapy. Immunohistochemistry to detect PD-L1 reactivity in tumors can be challenging, and additional methods are needed to predict and confirm PD-L1 expression. Here, we hypothesized that HNSCC tumor cell genomics influences cell signaling and downstream effects on immunosuppressive biomarkers and that these profiles can predict patient clinical responses. STUDY DESIGN: We identified deleterious gene mutations in SCC4, SCC15, and SCC25 and created cell line-specific predictive computational simulation models. The expression of 24 immunosuppressive biomarkers were then predicted and used to sort cell lines into those that would respond to PD-L1 immunotherapy and those that would not. RESULTS: SCC15 and SCC25 were identified as cell lines that would respond to PD-L1 immunotherapy treatment and SCC4 was identified as a cell line that would not likely respond to PD-L1 immunotherapy treatment. CONCLUSIONS: This approach, when applied to HNSCC cells, has the ability to predict PD-L1 expression and predict PD-1- or PD-L1-targeted treatment responses in these patients.

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.

Diminished Antimicrobial Peptide and Antifungal Antibiotic Activities against Candida albicans in Denture Adhesive.

The underlying causes of denture stomatitis may be related to the long-term use of adhesives, which may predispose individuals to oral candidiasis. In this study, we hypothesize that antimicrobial peptides and antifungal antibiotics have diminished anti-Candida activities in denture adhesive. To show this, nine antimicrobial peptides and five antifungal antibiotics with and without 1.0% denture adhesive were incubated with Candida albicans strains ATCC 64124 and HMV4C in radial diffusion assays. In gels with 1.0% adhesive, HNP-1, HBD2, HBD3, IP-10, LL37 (only one strain), histatin 5 (only one strain), lactoferricin B, and SMAP28 showed diminished activity against C. albicans. In gels with 1.0% adhesive, amphotericin B and chlorhexidine dihydrochloride were active against both strains of C. albicans. These results suggest that denture adhesive may inactivate innate immune mediators in the oral cavity increasing the risk of C. albicans infections, but inclusion of antifungal antibiotics to denture adhesive may aid in prevention or treatment of Candida infections and denture stomatitis.