John B. Matthews

Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease

Aims: To determine possible changes in gingival crevicular fluid (GCF) antioxidant defence in chronic adult periodontal disease and to investigate the nature of the local radical scavenging mechanisms, with particular reference to glutathione. Methods: GCF and plasma were collected from patients with chronic periodontitis and age and sex matched control subjects (n = 10). Polymorphonuclear leucocytes (PMNLs) were prepared and gingival epithelial cells (GECs) were collected by conventional methods from periodontally healthy subjects. PMNL were stimulated with F-Met-Leu-Phe after cytochalasin B treatment. Enhanced chemiluminescence was used to determine the total antioxidant capacity and to investigate the activity of cell fractions and reducing agents. GCF concentrations of reduced (GSH) and oxidised (GSSG) glutathione were determined by high performance liquid chromatography. Results: Plasma and GCF from patients contained lower mean (SD) total antioxidant capacity (501.8 (123) μM Teq/litre and 658.3 (392) μM Teq/litre, respectively) compared with controls (577.9 (99.8) and 1351.5 (861) μM Teq/litre, respectively). Antioxidant light recovery profiles for GCF demonstrated a stepped response, not seen in plasma, which was inhibited by N-ethylmaleimide. This response was also detected in the cytosolic fraction of GEC and anaerobically stimulated PMNL. Similar antioxidant profiles, inhibitable by N-ethylmaleimide, were obtained with cysteamine, cysteine, and GSH. Control GCF contained high mean (SD) concentrations of glutathione (GSH, 1899.8 (494.4)μM; GSSG, 256.8 (152.4)μM). GCF from patients with periodontitis contained significantly lower amounts of GSH (mean, 1183.1; SD, 580.3μM) and GSSG (mean, 150.1; SD, 44.9μM). Conclusions: GSH values and total antioxidant capacity are reduced in chronic periodontal disease. The high concentrations of GSH present in GCF in health are similar to those found extracellularly in the lung and may represent an important antioxidant and anti-inflammatory defence strategy common to exposed epithelial surfaces.

Enhanced chemiluminescent assay for measuring the total antioxidant capacity of serum, saliva and crevicular fluid.

This paper reports the development of an enhanced chemiluminescent (ECL) assay for measuring the total antioxidant (AO) capacity of serum, saliva and a fluid collectable from the gum margin called gingival crevicular fluid (GCF). The theory behind the assay is explained, and the optimum conditions for the assay, and for storage of reagents and clinical samples is described. Calibration lines were linear (R≥0·99; P<0·0001) and the within batch coefficient of variations for a water soluble vitamin E analogue (Trolox), serum and saliva samples were <5%. In saliva and GCF, a characteristic AO response not seen in serum of the same patients, was identified. Total peripheral (serum) and local (saliva) AO capacities (μmol/L Trolox) were investigated in patients with (n = 18) and without (n = 16) adult periodontitis. Serum AO status did not differ between groups. Salivary total AO concentrations were lower in the periodontitis (P) group [175 (53)μmol/L] than in the nonperiodontitis (NP) group [254 (110)μmol/L1: P<0·01], as were saliva:serum AO ratios [0·37 (0·11) versus 0·5 (0·18): P<0·01]. Periodontitis patients may have a reduced salivary AO concentration, which could result from, or predispose to, the damaging effects of reactive oxygen species (ROS). The potential for ROS production in the oral and periodontal environment may explain the presence of a specific antioxidant in oral fluids that is not detectable in serum. The ECL assay described provides a rapid, simple and reproducible method of measuring total antioxidant defence in small volumes of biological fluids.

Neutrophil Hyper-responsiveness in Periodontitis

Peripheral neutrophil hyper-responsiveness in chronic periodontitis leads to excessive reactive oxygen species (ROS) production. We aimed to determine whether neutrophil hyper-responsiveness was constitutive or reactive, and to discover the effect of non-surgical therapy. Peripheral blood neutrophils from patients (n = 19), before and 3 months after therapy, and matched control individuals were Fcγ-receptor-stimulated with/without priming with P. gingivalis and F. nucleatum. Total and extracellular ROS were determined by luminol/isoluminol chemiluminescence. The high total ROS generation of patients’ neutrophils compared with that of control individuals (P = 0.016) continued at a reduced level post-therapy (P = 0.059). Reduced activity post-therapy was also seen with priming. Unstimulated total ROS levels did not differ between patients and control individuals before or after therapy. However, the high unstimulated, extracellular ROS production by patients’ neutrophils compared with control individuals (P < 0.05) continued post-therapy and was unaffected by priming. Therapy reduced Fcγ-receptor-stimulated total ROS production, but not unstimulated extracellular radical release, suggesting that constitutive and reactive mechanisms underlie neutrophil hyper-responsiveness.

Differential activation of NF-κB and gene expression in oral epithelial cells by periodontal pathogens

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non‐viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll‐like receptors ‐2, ‐4 and ‐9, and components of the NF‐κB signalling pathway, immunocytochemical analyses were performed showing that NF‐κB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF‐κB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF‐κB pathway, including cytokines/chemokines TNF‐α, IL‐1β, IL‐8, MCP‐1/CCL2 and GM‐CSF, were up‐regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety‐one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase‐polymerase chain reaction (RT‐PCR) of molecules identified from the microarray data sets, including Heme oxygenase‐1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

Oral lichen planus: an immunoperoxidase study using monoclonal antibodies to lymphocyte subsets

Sixteen biopsies from patients with oral lichen planus (10), simple keratosis (3) and lichenoid reactions (3) were studied using monoclonal antibodies directed against lymphocyte markers. T lymphocytes predominated in the cellular infiltrates of the epithelium and lamina propria of all biopsies, and cells positive with Leu 3a and Leu 2a antibodies were also present at both sites in all specimens. However, many of the Leu 3a positive intraepithelial cells had a dendritic appearance and distribution consistent with their being Langerhans cells. Limited results from cell counts on three lichen planus specimens suggested that the majority of intraepithelial T cells were of the cytotoxic/suppressor phenotype (OKT3 and Leu 2a positive).

Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 in unicystic ameloblastoma

The expression of proliferating cell nuclear antigen (PCNA) and Ki‐67 was studied in unicystic and solid ameloblastoma (follicular and plexiform types) using a biotin‐streptavidin method on routinely processed paraffin sections. To determine percentage PCNA and Ki‐67 labelling indices, positive tumour cells and total tumour cells were counted in areas of each unicystic ameloblastoma corresponding to cystic linings, intraluminal nodules and invading tumour islands, and in solid ameloblastomas. Positive cells in basal and suprabasal layers of cystic tumour lining were also counted with respect to the length of basement membrane determined by image analysis. In unicystic ameloblastoma the invading islands exhibited a significantly higher PCNA labelling index (29.2 ± 16.4%) than intraluminal nodules (13.6 ± 5.4%; P < 0.05). Cystic tumour lining had relatively few PCNA positive cells and a labelling index (5.5 ± 3.3%) significantly lower than invading islands (P < 0.001) or intraluminal nodules (P < 0.003). The labelling indices of solid ameloblastomas of follicular type (48.1 ± 12.9%) were significantly higher than those of cystic tumour lining (P < 0.0001), intraluminal nodules (P < 0.001) and invading islands (P < 0.04) in unicystic ameloblastoma. Similar relationships were found for Ki‐67 expression except that comparisons involving invading islands and intraluminal nodules were not significant, a finding probably due to the smaller number of specimens available for quantitative analysis. These results indicate differences in proliferative potential between different areas of unicystic ameloblastoma and between unicystic and solid lesions. The fact that invading tumour islands within the fibrous tissue wall showed high labelling indices is in agreement with the clinical observation that their presence may be related to recurrence after conservative surgery. This provides a biological basis for indicating more radical surgical excision as the treatment of choice for this subgroup of lesions.

Trans-dentinal stimulation of tertiary dentinogenesis.

Trans-dentinal stimulation of tertiary dentinogenesis has long been recognized, and has traditionally been ascribed to diffusion of irritant substances arising during injury and restorative treatment. Identification of bio-active components, especially growth factors including TGF-βs, sequestered within dentin matrix provides a new explanation for cellular signaling during tertiary dentinogenesis. Both isolated dentin matrix components and pure growth factors (TGF-βs) have been shown to signal cellular events leading to reactionary and reparative tertiary dentinogenesis. Release of these bio-active components from dentin matrix may arise during carious attack and other injury to the tissue, and also during subsequent surgical intervention and restoration of the tooth. Both cavity-conditioning agents and leaching from restorative materials may contribute to release of these components. Distance of diffusion, as determined by cavity residual dentin thickness, and other restorative parameters may influence the signaling process after release of these components. Careful consideration of the interplay between tissue injury and surgical and restorative material factors is required for optimum exploitation of the exquisite regenerative capacity of dentin-pulp for more biological approaches to clinical treatment of dental disease.

Transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth.

Transforming growth factor (TGF)-β isoforms have been implicated in cellular signalling during tooth development and repair, but little is known of their cellular localisation or distribution within the dental tissues in the mature tooth. This study investigated the presence of TGF-β1, β2 and β3 isoforms in tissues of sound and carious human molar teeth, to understand better the expression of TGF-βs during health and disease. In healthy tissues, odontoblasts, cells of the cell rich layer, pulpal fibroblasts and endothelial cells were stained to varying degrees for all isoforms, with TGF-β3 showing the greatest intensity and TGF-β1 the weakest intensity. Similar patterns of staining were observed in carious teeth; however, TGF-β1 showed significantly increased staining intensity within odontoblasts and pulpal cells of carious teeth (p<0.001). Biochemical analysis showed greater amounts of TGF-β1 in tertiary dentine than in primary dentine samples. The expression of TGF-βs in odontoblasts and the increased presence of TGF-β1 in tertiary dentine suggest that these isoforms may be important in odontoblast behaviour and the modulation of the tissue response to injury.

Expression of epidermal growth factor receptors by odontogenic jaw cysts

The expression of epidermal growth factor receptor (EGFr) by odontogenic epithelium was studied in odontogenic cysts (n=35), ameloblastoma (n=6), and periapical granulomas containing proliferating epithelial rests of Malassez (n=7) using a panel of monoclonal antibodies to EGFr (clone E30, F4 and C11) known to react with formalin-fixed, paraffin-embedded sections. Odontogenic epithelium in all specimens demonstrated immunoreactivity with all three antibodies. Clone E30 consistently gave the most intense, membrane located staining pattern of the three antibodies tested. Generally, staining of epithelial cells progressively diminished with movement away from the basal cell layers toward the most superficial layers of cystic lining or centre of epithelial rests and tumour islands. Developmental odontogenic cysts (odontogenic keratocysts,n= 13; dentigerous cysts,n=11) and ameloblastoma (follicular type,n=5; unicystic type,n=1) expressed a higher level of EGFr staining than inflammatory cysts (radicular cysts,n=11) and the proliferating epithelial rests in periapical granulomas. However, foci of weak EGFr staining of odontogenic keratocyst lining, similar to that seen in radicular cysts, were found in areas associated with inflammation. In addition, epithelial rests not associated with inflammatory cell infiltrates exhibited stronger reactivity for EGFr than proliferating rests within periapical granulomas. These results indicate that the level of EGFr expression by odontogenic cysts and rests is related to the presence of inflammation within adjacent connective tissue and that there is no detectable difference in receptor expression between developmental cysts and ameloblastoma.

Decreased expression of TGF‐β cell surface receptors during progression of human oral squamous cell carcinoma

This study examined the immunocytochemical expression of the transforming growth factor‐β (TGF‐β) isoforms TGF‐β1, TGF‐β2, and TGF‐β3, together with the TGF‐β cell surface receptors TβR‐I and TβR‐II, in patient‐matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF‐β isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TβR‐II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TβR‐I, but not at a statistically significant level. This study also examined the structure of TβR‐I and TβR‐II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF‐β1. No structural abnormalities of TβR‐II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TβR‐II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709–718, codons 125–128), predicting a truncated receptor having no kinase domain. No defects were present in TβR‐I. The structures of TβR‐I and TβR‐II did not correlate with growth inhibition by TGF‐β1. The data suggest that decreased expression of TGF‐β receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TβR‐II expression, a dominant‐negative TβR‐II construct (dnTβR‐II) was transfected into a human oral carcinoma cell line with a normal TGF‐β receptor profile and known to be markedly inhibited by TGF‐β1. In those clones that overexpressed the dnTβR‐II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra‐1 and collagenase‐1 remained unchanged following treatment with TGF‐β1. The results demonstrate that a decrease in TβR‐II relative to TβR‐I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP‐1‐dependent genes that are involved in the regulation of the extracellular matrix. Copyright