Quantitative analysis of lymphocytes and their subsets in periapical lesions

The relative proportions of lymphocytes and their subsets in periapical lesions associated with untreated teeth and endodontically treated teeth were compared. Nine periapical lesions associated with previously untreated teeth and six persistent lesions associated with previously root treated teeth which had good quality root canal fillings and coronal seals were selected from a total of 41 samples. Clinical and radiographic features of each lesion were recorded. Serial frozen sections were stained using immunohistochemical methods and monoclonal antibodies against CD20, CD3, CD4 and CD8 to identify B, T, T helper (Th) and T suppressor (Ts) lymphocytes, respectively. Ten microscopic fields, representing the most dense cellular inflammatory infiltrate were selected per specimen and cell numbers were estimated, as a proportion of lesion area, using a point counting method. The periapical lesions associated with untreated teeth had a denser inflammatory cell infiltrate. The proportion of total lymphocytes was significantly higher in the untreated group of lesions ( P < 0.01). The proportions of B ( P < 0.01), T ( P < 0.001) and Th cells ( P < 0.01) were significantly higher in untreated teeth. The ratios of Th/Ts cells ( P < 0.05) and T/total lymphocytes ( P < 0.01) were also significantly different between the two types of lesion. The results show a difference in the inflammatory cell infiltrate and relative proportions of T, B and T helper cells in the two groups of lesions. This indicates that future studies of periapical lesions should take into account the clinical history of the associated teeth.     

Phenotypical and functional analysis of T cells in periodontitis

To explore aspects of cellular immune responses in the pathogenesis of periodontitis we analyzed phenotype and function of peripheral T cells. Two groups of subjects participated: one group consisted of 10 highly susceptible patients with severe periodontitis (mean age 29 years) and a control group consisted of 10 age, gender and race matched subjects with gingivitis. From all subjects peripheral blood was collected. The results showed that the numbers of CD3+, CD4+ and CD8+ T cells as well as the CD4/CD8 ratio, and the proliferative capacity of T cells, were not different between the two groups of subjects. Also, proportions of naive and memory T cells for both the CD4+ and CD8+ subpopulations were not different. Functional heterogeneity within the CD4+ and CD8+ T cell compartments was determined by intracellular analysis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production. On the basis of these latter analyses among CD4+ and CD8+ cells, T helper (Th) 1 or Th2 function and T cytotoxic (Tc) 1 or Tc2 function, respectively, could be deduced. No significant differences in proportions of CD4+ and CD8+ T cells positive for intracellular IFN-gamma or IL-4 were observed between periodontitis patients and gingivitis controls; however a higher level of intracellular IL-4 in CD8+ T cells was seen in periodontitis patients. This might indicate that there is a shift towards a Tc2 function within the CD8+ T cell subpopulation. The current explorative study suggests that further research into the role of CD8+ T cells in the pathogenesis of periodontitis is warranted.     

Crevicular fluid and serum IgG subclasses and corresponding mRNA expressing plasma cells in periodontitis lesions

Recent reports suggest that specific serum IgG subclasses are a feature of several forms of periodontitis. GCF antibodies are both serum-derived and locally produced by the abundant plasma cells of the diseased periodontal tissue. Previous work has shown that crevicular fluid (GCF) levels of IgG may be reduced in active and deep periodontal pockets when compared to other sites in chronic periodontitis patients (7). These findings, and more recent findings for IgA levels in GCF (5), suggest that GCF immunoglobulins may indicate "high risk" sites for periodontitis. In these studies, the relative distribution of IgG isotypes was not investigated, nor was the relative contribution of local and serum antibodies to the GCF immunoglobulin profile. Therefore, more precise investigation of the tissue distribution of local gingival IgG subclass producing plasma cells and their protein levels in the GCF from the same sites and in serum, was undertaken.     

Immunohistochemical localization of oncostatin M in epithelialized apical periodontitis lesions

Aim  To investigate the in situ location of oncostatin M (OSM) in epithelialized apical periodontitis lesions.
Methodology  Thirty periapical lesions of pulpal origin were collected with informed consent from patients at the time of apical surgery. Tissue specimens were fixed with 10% buffered formalin overnight, dehydrated in an ascending series of graded alcohol, and embedded in paraffin. Five micron sections from formalin-fixed, paraffin-embedded specimens were examined by immunohistochemistry. In addition, another section from each specimen was stained with haematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in OSM expression between tissue with low and high levels of inflammation were subsequently analyzed by Fisher's exact test.
Results  Based on histological examination of haematoxylin and eosin stained sections, all specimens revealed the morphology of epithelialized apical periodontitis lesions. The results from immunohistochemistry demonstrated that OSM stain was detected in the inflammatory infiltrates, epithelium, connective tissue, and endothelium. The OSM signal was mainly expressed in endothelial cells (100%) followed by inflammatory cells (93.33%), epithelial cells (53.33%), and fibroblasts (16.67%). In addition, OSM expression was significantly higher in epithelialized apical periodontitis with higher levels of inflammatory infiltrates ( P  < 0.001).
Conclusions  Oncostatin M was found to be expressed in epithelialized apical periodontitis lesions and would form part of the cytokine network involved in the disease process of apical periodontitis.     

Cathepsin B- and L-like activities at local gingival sites of chronic periodontitis patients

The cysteine proteinases cathepsins B and L have the potential to degrade connective tissue in chronic periodontitis and this may progress episodically at individual tooth sites. The activities of cathepsin B- and L-like proteinases in homogenised gingival tissue from control and periodontitis patients were measured biochemically using the selective peptide substrate Z-Phe-Arg-AFC and the selective cathepsin L inhibitor Z-Phe-Phe-CHN2. Each tooth site was divided, where appropriate, into gingival tissue and granulomata. These were assayed separately and the measurements related to the DNA and protein contents of the tissues. Enzyme activity in healthy control tissue was significantly lower than in diseased tissue. Enzyme activity in gingival tissue and total tissue from periodontitis patients decreased with increasing pocket depth, clinical attachment level, gingival index and bleeding index whilst cathepsin B activity in granulomata increased with increasing pocket depth and clinical attachment level but not with increasing gingival index or gingival bleeding index. Mean enzyme activity in gingival tissue was 1.6-2.8 times greater than in granulomata. Mean patient enzyme activity in diseased patients did not correlate positively with their mean pocket depth, clinical attachment level, gingival index or gingival bleeding index. These results are best explained by the probable cellular origins of the enzymes and the likely influence of their serum and tissue inhibitors during the disease process.     

自身免疫及其与牙周炎的关系

牙周炎是微生物引起的炎症性疾病,表现为局部牙周组织过度的免疫性炎症破坏。自身免疫可能参与了牙周炎病变过程中复杂的宿主免疫调控反应,其相关机制包括B-1细胞的活化、牙周组织自身抗体的存在、自身反应性T细胞的产生以及牙周致病菌的免疫刺激作用。本文就自身免疫性疾病产生的可能机制、自身免疫与牙周炎的关系作一综述。     

Immunoglobulin isotype distribution of locally produced autoantibodies to collagen type I in adult periodontitis

Production of antibodies to collagen type I was analyzed by means of an enzyme-linked immunospot (ELISPOT) assay in patients with chronic adult periodontitis (AP) before and after periodontal hygiene treatment. Anti-collagen type I antibody-secreting cells were found among mononuclear cells enzymatically eluted from inflamed gingiva in 9 of 15 patients with untreated AP and in 4 of 14 hygiene-treated patients with a varied isotype distribution. A notably high prevalence of IgG and IgM isotypes was observed for the anti-collagen antibodies in untreated patients. With wide variation, chronic AP was characterized by a high frequency of spontaneous IgG and low numbers of IgA and IgM-producing cells. Periodontal hygiene treatment significantly reduced the number of IgA and IgM-secreting cells. Although AP is not an autoimmune disease in the accepted sense, our results indicate that local autoimmune reactions to collagen type I are common in untreated AP, implying an interplay between periodontal infection and autoimmunity.     

Endothelial cell leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression in gingival tissue during health and experimentally-induced gingivitis

The changes in vascular adhesion molecule expression and numbers of infiltrating leukocytes during a 21-day experimental gingivitis episode were investigated immunohistochemically. Monoclonal antibodies to ELAM-1 (1.2B6), ICAM-1 (6.5B5), CD3 (OKT3-pan-T cell) and neutrophils (PMN-elastase) were used to identify positive vessels and leukocytes within gingival biopsies taken on d 0, 7, 14 and 21. Vascular endothelium expressed ELAM-1 and ICAM-1 both in clinically 'healthy' tissue (d 0) and in experimentally inflamed tissue (d 7 to 21). Positive vessels were found mainly in the connective tissue subjacent to the junctional epithelium where the highest numbers of T cells and neutrophils were also seen. Although T cells were found in all tissue areas studied, neutrophils were largely concentrated in the junctional epithelium and the subjacent connective tissue but were absent from the oral epithelial region. As the experimental gingivitis developed, the number of T cells or neutrophils in the different tissue regions did not change significantly although the most intense vascular ICAM-1 and ELAM-1 staining redistributed to the CT adjacent to the junctional epithelium. A prominent feature was the intense ICAM-1 positive staining of the junctional epithelium and its absence in the closely adjacent oral epithelium, in both clinically 'healthy' and inflamed tissue. The gradient of ICAM-1 in junctional epithelium, with the strongest staining on the crevicular aspect plus the vascular expression of ELAM-1 and ICAM-1 in both clinically 'healthy' and inflamed tissue may be crucial processes which direct leukocyte migration towards the gingival crevice.     

The distribution of plasma cells in naturally occurring periodontitis lesions in rats. A light and electron microscopic study

Plasma cells are widely distributed in inflamed periodontal tissues, the adjacent periodontal ligament and nearby alveolar bone spaces, of old rats (20 and 27 months) raised on a conventional diet in a normal laboratory environment. Electron microscopy revealed three morphologic types (or perhaps stages) of these cells based on the arrangement and content of the granular endoplasmic reticulum. Close contact between plasma cells and other cell types, such as lymphocytes, macrophages and fibroblasts, were commonly observed. It is suggested that plasma cell infiltration is a widespread and prominent feature of naturally occurring periodontitis in old rats, resembling the condition known to exist in humans, monkeys and dogs. The occurrence of large numbers of apparently fully differentiated plasma cells in otherwise normal alveolar bone marrow is discussed.     

Expression of beta 1 integrins in human dental pulp in vivo: a comparative immunohistochemical study on healthy and chronic marginal periodontitis samples

AIM: The objective of this study was to determine the tissue distribution of beta 1 integrin chains in sound human dental pulps and to compare the findings with connective tissue compartments of other organs and to pulp tissue in teeth extracted due to periodontal disease. METHODOLOGY: Freshly frozen pulp tissue samples from teeth extracted for orthodontic reasons were examined and compared to samples from teeth extracted due to chronic (marginal) periodontitis. beta 1 integrin chains were determined using an indirect-immunoperoxidase technique. Seven monoclonal antibodies recognizing alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 1 chains of Very Late Activation Antigen (VLA) integrins were used for this purpose. RESULTS: VLA-1, VLA-2, VLA-3 and VLA-5 were expressed by vascular endothelium and vascular smooth muscle in varying intensities in both groups. VLA-6 reactivity was observed in the basal surfaces of arterial, venous and capillary endothelia. Our results indicate that there was no significant difference in the expression of VLA integrins in sound pulp tissue when compared to the samples from chronic (marginal) periodontitis and the connective tissue compartments of other viscera. CONCLUSION: The present findings suggest that human dental pulp tissue is not different from other connective tissue compartments in the body with respect to VLA integrin expression, and chronic marginal periodontitis does not affect pulp tissue to a histopathologically detectable extent.     

Salivary and plasma levels of Toll-like receptor 2 and Toll-like receptor 4 in chronic periodontitis

Background: This cross‐sectional study was planned to investigate whether patients with chronic periodontitis exhibit different salivary or plasma concentrations of Toll‐like receptor (TLR) 2 and TLR4 compared to subjects who are clinically healthy. Methods: Whole saliva and plasma samples were obtained and full‐mouth clinical periodontal measurements were recorded from 22 otherwise healthy patients with chronic periodontitis and 21 systemically and periodontally healthy control subjects. Salivary and plasma TLR2 and TLR4 levels were determined by enzyme‐linked immunoassays. Data were tested statistically using Mann‐Whitney U test. Results: The healthy group exhibited significantly lower values in all clinical measurements (P <0.001). The salivary TLR2 levels were similar in the two study groups (P >0.05). The patients with chronic periodontitis exhibited significantly higher salivary TLR4 (P <0.01) and plasma TLR2 and TLR4 levels (P <0.05). Conclusion: The present findings support a hypothesis that inflammation increases expression of TLRs which leads to an increased detection of TLRs in saliva and plasma, which could be useful as a diagnostic test for periodontal diseases.     

Some cytokine profiles of T-helper cells in lesions of advanced periodontitis

OBJECTIVE: The aim of the present study was to analyze some cytokine profiles of T-helper cells in periodontitis lesions. MATERIAL AND METHODS: 22 adult patients (7 females and 15 males, aged 24-66 years) with advanced and generalized chronic periodontitis were recruited. Clinical and radiographical characteristics of periodontal disease was assessed. From each patient a gingival biopsy was obtained from one randomly selected diseased interproximal site. The soft tissue sample was prepared for immunohistochemical analysis. Double staining was performed to detect cells positive for both the CD4 marker and different cytokines, i.e. interleukin (IL)-2, IL-4, IL-6 and interferon-gamma (IFN-gamma). RESULTS: The lesions in advanced periodontitis contained similar proportions of cells positive for the different cytokine markers examined. In addition, the number of cells expressing cytokine profiles for either T helper-1 (IFN-gamma + IL-2) or T helper-2 (IL-4 + IL-6) was similar. CONCLUSION: It is suggested that the lesions of periodontitis are regulated by a combined Th-1 and Th-2 function.