David F. Lappin

Cytokine Responses of Oral Epithelial Cells to Porphyromonas gingivalis Infection

Accumulating evidence indicates that epithelia are not merely mechanical barriers but also important elements of the innate immune system. The present study was performed to examine cytokine responses of oral epithelial cells after infection with the periodontal pathogen Porphyromonas gingivalis. The KB-cell line and primary cultures of periodontal pocket epithelium were infected with P. gingivalis for assessment of bacterial invasion by an antibiotic protection assay, and examinination of expression of interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor-alpha by in situ hybridization and immunohistochemistry. We observed that P. gingivalis induces a strong cytokine response, positively correlated with the adhesive/invasive potential of the infecting strain, in both KB cells and primary cultures. These findings indicate that the epithelial cells of the periodontal pocket are an integral part of the immune system, eliciting cytokine responses to a bacterial challenge. In this context, the adhesive/invasive phenotype of P. gingivalis appears to contribute to pathogenicity.

Anti-inflammatory cytokine IL-10 and T cell cytokine profile in periodontitis granulation tissue

Th2 cells are more abundant than Th1 cells in periodontitis lesions, but the relative importance of the Th1 and Th2 subsets in periodontal disease is not understood. In addition, the role of proinflammatory and anti‐inflammatory cytokines in this disease process is unclear. Biopsies were obtained from 10 patients with early onset periodontitis (EOP) and 10 patients with adult periodontitis (AP). From all of the patients in the AP group we were able to obtain and section the gingival tissue to serve as controls. We used polyclonal monospecific antibodies to detect cells expressing IL‐2, IL‐4, IL‐6, IL‐10 and IL‐15, tumour necrosis factor (TNF‐α) and interferon‐gamma (IFN‐γ) in formalin‐fixed, paraffin‐embedded sections of granulation tissue from periodontitis lesions. We also employed a series of oligonucleotide probes to detect cells expressing the cytokine transcripts in the same tissue biopsies. Cells that expressed IL‐4 or IL‐6 were more numerous than cells expressing either IL‐2 or IFN‐γ. Th2 cells were more numerous in EOP and AP tissues. IL‐15 substitutes for IL‐2 in a number of biological activities related to the Th1 immune response, and interestingly, in periodontal lesions the IL‐15‐expressing cells outnumbered IL‐2‐expressing cells, suggesting that this is the pattern of immune regulation by T cells in the periodontium. The functional balance in the T cell subsets detected by their cytokine profiles underlies the importance of the anti‐inflammatory mechanisms taking place in the diseased tissue. The numbers of inflammatory leucocytes that express the anti‐inflammatory cytokine IL‐10 are much more widely distributed than those that express the proinflammatory cytokines IL‐6 and TNF‐α. This study suggests that large numbers of infiltrating inflammatory cells as well as accessory cells are involved in the down‐regulation of the inflammatory and immune response in periodontitis.

Clinical, pathological and immunological aspects of periodontal disease

The inflammatory and immune responses during the development and progression of periodontitis are reviewed. Susceptibility to periodontitis may be related to whether plasma cells predominate in the tissues of an individual, or a site, in response to the microbial insult from dental plaque. The tendency for an individual or site to form an extensive plasma cell infiltrate may indicate an inability to defend against periodontopathogenic bacteria and thus a predisposition to periodontitis. Selected pertinent areas of current interest in cellular and humoral immunology are considered within the periodontal context. These topical issues include (a) homing of immune and inflammatory cells to target tissues; (b) the local proliferation and synthetic activity of immune and inflammatory cells; (c) the cytokine profile of the inflammatory and immune cells; and (d) the immunoglobulin subclasses of locally produced antibodies.

Azole Resistance of Aspergillus fumigatus Biofilms Is Partly Associated with Efflux Pump Activity

ABSTRACT This study investigated the phase-dependent expression and activity of efflux pumps in Aspergillus fumigatus treated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P < 0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P < 0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for the A. fumigatus germling cells by 2- to 8-fold. Quantitative expression analysis of AfuMDR4 mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P < 0.001). Finally, an in vivo mouse biofilm model was used to evaluate efflux pump expression, and it was shown that AfuMDR4 was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P < 0.01). Our results demonstrate that efflux pumps are expressed in complex A. fumigatus biofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis.

Gingival epithelial cells heterozygous for Toll-like receptor 4 polymorphisms Asp299Gly and Thr399Ile are hypo-responsive to Porphyromonas gingivalis

The Toll-like receptor (TLR)4 is the major sensor for bacterial lipopolysaccharide and its two common co-segregating polymorphisms, Asp299Gly and Thr399Ile, which occur at a frequency of between 6 and 10%, have been associated with infectious diseases, LPS hypo-responsiveness and cardiovascular disease. Porphyromonas gingivalis is a Gram-negative bacterium implicated in chronic periodontitis and is a known TLR4 and TLR2 agonist. We obtained two gingival epithelial cell primary cultures from subjects heterozygous for the TLR4 polymorphism Asp299Gly and compared response characteristics with similar cells from patients (four) with the wild-type TLR4 genes. Cytokine responses and transcriptome profiles of gingival epithelial cell primary culture cells to TNFα challenge were similar for all primary epithelial cell cultures. P. gingivalis challenge, however, gave markedly different responses for Asp299Gly heterozygous and wild-type epithelial cell cultures. The epithelial cells heterozygous for the TLR4 polymorphism Asp299Gly were functionally hypo-responsive, evidenced by differences in BD-2 mRNA expression, mRNA response profile by microarray analysis and by pro-inflammatory and chemokine cytokines at the protein and mRNA level. These findings emphasize variance in human epithelial cell TLRs, linked with Asp299Gly carriage, which results in a hypo-responsive epithelial cell phenotype less susceptible to Gram-negative diseases and associated systemic conditions.

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

ABSTRACT Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.

Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.

BACKGROUND Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals. AIM To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres. METHODS Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA. RESULTS Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean ± SEM: 1.37 ± 0.23 versus 0.40 ± 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 ± 4385 versus 2512 ± 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 ± 0.35; non-smokers: 1.41 ± 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 ± 0.19 AU), at levels between healthy non-smokers (0.15 ± 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01). CONCLUSION In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

Biofilm formation by Scottish clinical isolates of Staphylococcus aureus

The biofilm-forming capacity of 972 clinical isolates of Staphylococcus aureus was tested using a high-throughput polystyrene 96-peg plate format. Isolates of S. aureus were collected from patients in hospitals throughout Scotland from 2004 to 2006; 763 of these were meticillin-resistant S. aureus (MRSA) and 209 were meticillin-sensitive S. aureus (MSSA). The biomass of each biofilm was quantified using a crystal violet staining technique. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The majority of MRSA (53.8 %) and MSSA (43.5 %) isolates formed moderately attached biofilms. Fully established biofilms were formed by 20.5 % of MRSA isolates and 28.0 % of MSSA isolates, whilst 25.7 % of MRSA isolates and 28.5 % of MSSA isolates formed negligible biofilms. There was no significant correlation between susceptibility to meticillin and biofilm formation (P=0.77). MRSA isolates were divided into clonal types (EMRSA-15, EMRSA-16 and sporadic isolates) based on PFGE genotyping results. EMRSA-15 isolates formed significantly more moderately and fully established biofilms than EMRSA-16 isolates (P<0.001). S. aureus strains isolated from the skin of patients had a significantly greater capacity to form biofilms than isolates from other body sites, including the blood. Microscopic examination of biofilms by scanning electron microscopy (SEM) revealed that poorly adherent biofilm formers failed to colonize the entire surface of the peg, whilst moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group that formed fully established biofilms confirmed that this isolate developed a dense biofilm with a textured, multi-layered, three-dimensional structure.

Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm

The Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins. Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells. We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter. After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies. Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA. When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained. However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi. In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi. Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter. The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm. These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.

Saliva concentrations of RANKL and osteoprotegerin in smoker versus non-smoker chronic periodontitis patients

OBJECTIVES To compare the salivary receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) concentrations in smokers versus non-smokers with chronic periodontitis. MATERIAL AND METHODS Whole saliva samples were obtained from 67 untreated chronic periodontitis patients, of whom 34 were smokers, and from 44 maintenance patients, of whom 22 were smokers. Full-mouth clinical periodontal measurements were recorded. Saliva cotinine, sRANKL and OPG concentrations were determined by ELISA. Statistical analysis was performed using the Mann-Whitney U test, Bonferronis correction for multiple comparisons and Spearmans correlations. RESULTS Untreated smokers exhibited significantly higher values of clinical periodontal recordings than untreated non-smokers (all p<0.05). Salivary cotinine level correlated with clinical attachment level (p=0.023). Smoker versus non-smoker maintenance groups showed no significant differences in clinical parameters. There were significant differences in sRANKL and OPG concentrations between untreated and maintenance groups (all p<0.01). Salivary OPG concentration was significantly lower (all p<0.01) and the sRANKL/OPG ratio was higher (all p<0.01) in smokers than in non-smokers. OPG concentration correlated positively with probing depth, clinical attachment level and bleeding on probing (all p<0.005) and negatively with pack-year, and cotinine level (p<0.05). CONCLUSION Salivary RANKL and OPG concentrations are suggested to be affected by smoking as not only the untreated but also the treated smokers exhibited higher RANKL and lower OPG concentrations than non-smokers.