Amber M. Bates

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

Significance Middle East respiratory syndrome, caused by a zoonotically transmitted coronavirus (MERS-CoV), has a high mortality (∼36%). Because of limited autopsy data on tissues from MERS fatalities, a small animal model can provide an important tool to better understand the disease. We humanized the mouse locus of the virus receptor DPP4, preserving native DPP4 expression. After inoculating hDPP4 knockin mice with MERS-CoV, there was virus replication without disease. We then generated a mouse-adapted MERS-CoV by serial passage in hDPP4 knockin mice. The resultant virus causes fatal lung disease that includes diffuse alveolar damage and immune dysregulation. Here, we characterize the pathologic features of the model and elucidate key aspects of the immunopathology and factors contributing to virulence. 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.

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 μM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propidium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2-10.0 μM long-chain bases and GML were not cytotoxic; 40.0-80.0 μM long-chain bases, but not GML, were cytotoxic; and 80.0 μM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

Antimicrobial Activity of Chemokine CXCL10 for Dermal and Oral Microorganisms

CXCL10 (IP-10) is a small 10 kDa chemokine with antimicrobial activity. It is induced by IFN-γ, chemoattracts mononuclear cells, and promotes adhesion of T cells. Recently, we detected CXCL10 on the surface of the skin and in the oral cavity. In the current study, we used broth microdilution and radial diffusion assays to show that CXCL10 inhibits the growth of Escherichia coli, Staphylococcus aureus, Corynebacterium jeikeium, Corynebacterium striatum, and Candida albicans HMV4C, but not Corynebacterium bovis, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Poryphromonas gingivalis, or C. albicans ATCC 64124. The reason for the selective antimicrobial activity is not yet known. However, antimicrobial activity of CXCL10 may be related to its composition and structure, as a cationic 98 amino acid residue molecule with 10 lysine residues, 7 arginine residues, a total net charge of +11, and a theoretical pI of 9.93. Modeling studies revealed that CXCL10 contains an α-helix at the N-terminal, three anti-parallel β-strands in the middle, and an α-helix at the C-terminal. Thus, CXCL10, when produced on the surface of the skin or in the oral cavity, likely has antimicrobial activity and may enhance innate antimicrobial and cellular responses to the presence of select commensal or opportunistic microorganisms.

Predicting PD-L1 expression on human cancer cells using next-generation sequencing information in computational simulation models

PurposeInteraction of the programmed death-1 (PD-1) co-receptor on T cells with the programmed death-ligand 1 (PD-L1) on tumor cells can lead to immunosuppression, a key event in the pathogenesis of many tumors. Thus, determining the amount of PD-L1 in tumors by immunohistochemistry (IHC) is important as both a diagnostic aid and a clinical predictor of immunotherapy treatment success. Because IHC reactivity can vary, we developed computational simulation models to accurately predict PD-L1 expression as a complementary assay to affirm IHC reactivity.MethodsMultiple myeloma (MM) and oral squamous cell carcinoma (SCC) cell lines were modeled as examples of our approach. Non-transformed cell models were first simulated to establish non-tumorigenic control baselines. Cell line genomic aberration profiles, from next-generation sequencing (NGS) information for MM.1S, U266B1, SCC4, SCC15, and SCC25 cell lines, were introduced into the workflow to create cancer cell line-specific simulation models. Percentage changes of PD-L1 expression with respect to control baselines were determined and verified against observed PD-L1 expression by ELISA, IHC, and flow cytometry on the same cells grown in culture.ResultThe observed PD-L1 expression matched the predicted PD-L1 expression for MM.1S, U266B1, SCC4, SCC15, and SCC25 cell lines and clearly demonstrated that cell genomics play an integral role by influencing cell signaling and downstream effects on PD-L1 expression.ConclusionThis concept can easily be extended to cancer patient cells where an accurate method to predict PD-L1 expression would affirm IHC results and improve its potential as a biomarker and a clinical predictor of treatment success.

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.

Antimicrobial Peptides in Host Defense: Functions Beyond Antimicrobial Activity

Antimicrobial peptides are well known for their important roles in host defense by enhancing the barrier function and limiting microbial populations of the skin and mucosa. However, many of these peptides are now known to have additional roles assisting innate and adaptive immune functions. To facilitate innate immunity, antimicrobial peptides activate complement, chemoattract cells (e.g., monocytes, macrophages, T cells, neutrophils, immature dendritic cells, and mast cells), enhance phagocytosis, and modulate the production of chemokines and proinflammatory cytokines in other cells. At local sites of initiation, antimicrobial peptides can act as opsonins to enhance phagocytosis by monocytes and phagocytes and can activate cells. In the latter, for example, treatment of osteoblasts and osteoblast-like MG63 cells with human beta-defensin (HBD)2 increases their proliferation rates. Treatment of osteoblast-like MG63 cells with HBD2 and HBD3 increases transcript levels of osteogenic markers for differentiation, increases antileukoprotease (ALP) levels, and enhances mineralized nodule formation. To facilitate adaptive immunity, antimicrobial peptides assist the uptake of antigens by monocytes or other antigen-presenting cells and later direct the process toward a Th1 or Th2 adaptive immune response. More commonly though, antimicrobial peptides induce a mixed response characterized by Th1-/Th2-specific antibodies and Th1/Th2 cytokines from antigen-exposed splenocytes of immunized animals. Finally, antimicrobial peptides can be detected in the margins around both oral and cutaneous wounds, and there is growing evidence to suggest they also play a dynamic role in wound healing by improving wound angiogenesis, vascularization, and reepithelialization.

Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions

Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro.

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.