Shannon M. Wallet

Triclosan alters antimicrobial and inflammatory responses of epithelial cells

UNLABELLED Periodontal diseases are a class of pathologies wherein oral microbes induce harmful immune responses in a susceptible host. Therefore, an agent that can both reduce microbial burden and lessen pathogenesis of localized inflammation would have beneficial effects in periodontal disease; 2,4,4-trichloro-2-hydroxydiphenyl-ether [triclosan] is currently used in oral care products owing to broad spectrum antimicrobial and anti-inflammatory properties. OBJECTIVE To determine effects of triclosan on the response of oral epithelial cells to stimulation with the inflammatory microbial product lipopolysaccharide (LPS), a ligand for toll-like receptor 4 [TLR4]. MATERIALS/METHODS Primary human oral epithelial cells were stimulated with LPS in the presence and/or absence of triclosan after which expression of pro-inflammatory cytokines, β-defensins, micro-RNAs [miRNAs], or TLR-signaling pathway proteins were evaluated. RESULTS Here, we demonstrate that triclosan is a potent inhibitor of oral epithelial cell LPS-induced pro-inflammatory responses by inducing miRNA regulation of the TLR-signaling pathway. Triclosan was not a pan-suppresser of oral epithelial cell responses as β-defensin 2 [βD2] and βD3 were upregulated by triclosan following LPS-stimulation. CONCLUSIONS These data demonstrate both a novel antimicrobial mechanism by which triclosan improves plaque control and an additional anti-inflammatory property, which could have beneficial effects in periodontal disease resolution.

Enteric bacteria promote human and mouse norovirus infection of B cells

Bacteria help norovius infect B cells Stomach ache, nausea, diarrhea—many people know the sort of gastrointestinal havoc norovirus can wreak. Despite this, norovirus biology remains unclear, because human norovirus cannot be grown in culture. Jones et al. now report that with the help of bacteria, human norovirus can infect cultured B cells (see the Perspective by Robinson and Pfeiffer). To infect B cells, human norovirus required the presence of gut bacteria that expressed proteins involved in determining blood type. Mouse norovirus also infected B cells, and the treatment of mice with antibiotics protected them from norovirus infection. Science, this issue p. 755; see also p. 700 Gut bacteria that express histo-blood group antigens help human norovirus to infect B cells. [Also see Perspective by Robinson and Pfeiffer] The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual’s histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

Sepsis Induces Early Alterations in Innate Immunity That Impact Mortality to Secondary Infection

Sepsis, the systemic inflammatory response to microbial infection, induces changes in both innate and adaptive immunity that presumably lead to increased susceptibility to secondary infections, multiorgan failure, and death. Using a model of murine polymicrobial sepsis whose severity approximates human sepsis, we examined outcomes and defined requirements for survival after secondary Pseudomonas aeruginosa pneumonia or disseminated Listeria monocytogenes infection. We demonstrate that early after sepsis neutrophil numbers and function are decreased, whereas monocyte recruitment through the CCR2/MCP-1 pathway and function are enhanced. Consequently, lethality to Pseudomonas pneumonia is increased early but not late after induction of sepsis. In contrast, lethality to listeriosis, whose eradication is dependent upon monocyte/macrophage phagocytosis, is actually decreased both early and late after sepsis. Adaptive immunity plays little role in these secondary infectious responses. This study demonstrates that sepsis promotes selective early, impaired innate immune responses, primarily in neutrophils, that lead to a pathogen-specific, increased susceptibility to secondary infections.

Human norovirus culture in B cells

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

Bacterial Colonization of the Dental Implant Fixture-Abutment Interface: An In Vitro Study

BACKGROUND The geometry of the fixture-abutment interface (FAI) might influence the risk of bacterial invasion of the internal part of the implant. The aim of this study was to use an in vitro model to assess the potential risk for invasion of oral microorganisms into the FAI microgap of dental implants with different characteristics of the connection between the fixture and abutment. METHODS Thirty implants were divided into three groups (n = 10 per group) based on their microgap dynamics. Groups 1 and 2 were comprised of fixtures with internal Morse-taper connections that connected to standard abutments and the same abutments with a 0.5-mm groove modification, respectively. Group 3 was comprised of implants with a tri-channel internal connection. Fixtures and abutments were assembled and allowed to incubate in a bacterial solution of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis. Two standard abutments were either exposed to bacterial culture or left sterile to serve as positive and negative controls. After disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment, plated, and allowed to culture under appropriate conditions. RESULTS Three of the 10 samples in group 1 developed one colony forming unit (CFU) for A. actinomycetemcomitans, whereas zero of 10 samples developed CFUs for P. gingivalis. Ten of 10 and nine of 10 samples from groups 2 and 3, respectively, developed multiple CFUs for A. actinomycetemcomitans and P. gingivalis. CONCLUSION This study indicated that differences in implant designs may affect the potential risk for invasion of oral microorganisms into the FAI microgap.

Bacterial Colonization of the Implant–Abutment Interface Using an In Vitro Dynamic Loading Model

BACKGROUND Previously, we demonstrated that the geometry of the fixture-abutment interface influences the risk of bacterial invasion into the internal part of the implant, although the contribution of loading on this invasion was not evaluated. The aim of the present study is to use an in vitro dynamic-loading model to assess the potential risk for invasion of oral microorganisms into the fixture-abutment interface microgap of dental implants with different fixture-abutment connection characteristics. METHODS Twenty-eight implants were divided into two groups (n = 14 per group) based on their microgap dynamics. Group 1 was comprised of fixtures with internal Morse-taper connection that connected to standard abutments. Group 2 was comprised of implants with a four-groove conical internal connection that connected to multibase abutments. The specimens were immersed in a bacterial solution of Escherichia coli and loaded with 500,000 cycles of 15 N in a wear simulator. After disconnection of fixtures and abutments, microbial samples were taken from the threaded portion of the abutment, plated, and cultured under appropriate conditions. The difference between loosening and tightening torque value was also measured. RESULTS One of the 14 samples in Group 1 and 12 of the 14 of samples in Group 2 developed multiple colony forming units for E. coli. Implants in Group 1 exhibited an increase in torque value in contrast to implants in Group 2, which exhibited a decrease. CONCLUSION This study indicates that differences in implant design may affect the potential risk for invasion of oral microorganisms into the fixture-abutment interface microgap under dynamic-loading conditions.

Hyper-responsive Phenotype in Localized Aggressive Periodontitis

The ‘hyper-responsive’ trait is an increased inflammatory response upon stimulation of innate immune receptors. Our objective was to determine if a hyper-reactive trait is present in a cohort diagnosed with aggressive periodontitis (LAgP). Peripheral blood was collected from 30 LAgP, 10 healthy unrelated, and 10 healthy sibling participants and stimulated with lipopolysaccharide (LPS) from E. coli and P. gingivalis. Cyto/chemokine response profiles were evaluated and analyzed by ANOVA. Elevated levels of pro-inflammatory cyto/chemokines were detected in E. coli and P. gingivalis LPS-stimulated LAgP cultures when compared with those of healthy unrelated control individuals. Periodontally healthy siblings presented with attenuated hyper-inflammatory cyto/chemokine profiles. Regression analysis demonstrated the hyper-reactive trait to be concomitant expression of pro-inflammatory cyto/chemokines and an absence of anti-inflammatory mediator expression. Our findings demonstrate hyper-responsive trait in a LAgP cohort, along with an attenuated hyper-responsiveness in healthy siblings, which can be induced in response to multiple TLR ligations.

Local Inflammatory Markers and Systemic Endotoxin in Aggressive Periodontitis

While much research has focused on local and systemic factors contributing to periodontal disease, little is known regarding mechanisms linking these factors. We have previously reported a systemic hyper-inflammatory response to bacterial endotoxin in localized aggressive periodontitis (LAP). The objectives of this study were to delineate cyto/chemokines in gingival crevicular fluid (GCF) and evaluate systemic levels of endotoxin associated with LAP. Clinical parameters, GCF, and peripheral blood were collected from: 34 LAP, 10 healthy siblings, and nine healthy unrelated control individuals. Cyto/chemokines were quantified in GCF, systemic endotoxin levels were quantified in plasma, and correlation analysis was performed among all parameters. Nine mediators were elevated in LAP diseased sites as compared with healthy sites (TNFα, INFγ, IL1β, IL2, IL6, IL10, Il12p40, GMCSF, and MIP1α, p < 0.001), while MCP1, IL4, and IL8 were elevated in healthy sites (p < 0.01). Four- to five-fold-higher endotoxin levels were detected in LAP plasma compared with that from healthy participants (p < 0.0001), which correlated with all clinical parameters and most cyto/chemokines analyzed. In conclusion, higher systemic levels of endotoxin were found in LAP, which correlates with an exacerbated local inflammatory response and clinical signs of disease. (Clinicaltrials.gov number, NCT01330719).

Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling in THP-1 cells

GW bodies (GWB or P bodies) are cytoplasmic foci thought to result from microRNA (miRNA) regulation of messenger RNA (mRNA) targets and subsequent mRNA degradation. The purpose of this study is to examine the effects of lipopolysaccharide (LPS) stimulation of human monocytes on GWB formation, miRNA induction, miRNA target regulation and downstream cytokine and chemokine expression. In response to LPS stimulation, the number of GWB consistently increased by twofold at 8 h after stimulation and this increase was abolished when the miRNA‐effector proteins Rck/p54 or argonaute 2 were depleted. As the level of miR‐146a increased from 19‐fold up to 100‐fold during LPS stimulation, the transfection of a miR‐146a mimic into THP‐1 cells was examined to determine whether miR‐146a alone can induce similar changes in GWB. The results showed transfected miR‐146a could produce a comparable increase in the number of GWB and this was accompanied by a reduction in major cytokines/chemokines induced by LPS. These data show that the increase in size and number of GWB may serve as a biomarker for miRNA‐mediated gene regulation, and miR‐146a has a significant role in the regulation of LPS‐induced cytokine production in THP‐1 cells.

Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease

OBJECTIVE This study was designed to test the hypothesis that periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis are synergistic in terms of virulence potential using a model of mixed-microbial infection in rats. MATERIALS AND METHODS Three groups of rats were infected orally with either T. forsythia or P. gingivalis in mono-bacterial infections or as mixed-microbial infections for 12 weeks and a sham-infected group were used as a control. This study examined bacterial infection, inflammation, immunity, and alveolar bone loss changes with disease progression. RESULTS Tannerella forsythia and P. gingivalis genomic DNA was detected in microbial samples from infected rats by PCR indicating their colonization in the rat oral cavity. Primary infection induced significantly high IgG, IgG2b, IgG1, and IgG2a antibody levels indicating activation of mixed Th1 and Th2 immune responses. Rats infected with the mixed-microbial consortium exhibited significantly increased palatal horizontal and interproximal alveolar bone loss. Histological examinations indicated significant hyperplasia of the gingival epithelium with moderate inflammatory infiltration and apical migration of junctional epithelium. The results observed differ compared to uninfected controls. CONCLUSION Our results indicated that T. forsythia and P. gingivalis exhibit virulence, but not virulence synergy, resulting in the immuno-inflammatory responses and lack of humoral immune protection during periodontitis in rats.