Immunohistochemical analysis of inflammatory infiltrate in aggressive and chronic periodontitis: a comparative study

This immunohistochemical study evaluated the inflammatory infiltrate with its cluster differentiation markers (CD 4, CD 8, CD 20, and CD 68) in aggressive and chronic periodontitis gingival tissues in order to identify the specific cell distribution. Twenty-seven human gingival biopsies were obtained and analyzed. Fourteen patients were suffering from chronic periodontitis and six from aggressive periodontitis; seven patients with healthy gingiva were included as the control group. The specimens were immunohistochemically stained for anti-CD 4 (T helper cells), anti-CD 8 (T cytotoxic/suppressor), anti CD-20 (B plasma cells) and anti CD-68 (macrophages). Chronic periodontitis samples were mainly dominated by CD 4 and CD 8+ cells. On the contrary, in aggressive periodontitis patients all four cell types (CD 4, CD 8, CD 20 and CD 68 + cells, respectively) were remarkably increased. CD 20+ cells were significantly (p < 0.05) more prevalent in aggressive versus chronic periodontitis. The control samples expressed lower CD 4, CD 8, CD 20 and CD 68+ cells confirming a none inflammatory state. The present study demonstrates prevalence for CD 20+ cells in aggressive periodontitis lesions. However, further studies need to be performed to confirm and identify a clear pattern of inflammatory cells and hereafter the mechanisms sustaining the disease.     

In situ characterization of mononuclear cells in human chronic marginal periodontitis using monoclonal antibodies

Acetone fixed cryostat sections from 25 patients with adult chronic marginal periodontitis were characterized using an indirect immunofluorescence technique with monoclonal antibodies. The amount of B lymphocytes (Leu-12 positive) varied considerably between the specimens and were usually seen in largest numbers in the most apical parts of the cellular infiltrates beneath the pocket epithelium (PE). Varying amounts of T lymphocytes (OKT 3 positive) were demonstrated in all specimens. The amount of T helper cells (OKT 4a positive) exceeded that of T suppressor/cytotoxic cells (OKT 8 positive) in the cellular infiltrates beneath the PE (OKT 4a/ OKT 8 =1.13). There was a more even distribution of these cell types beneath the oral gingival epithelium (OGE). Langerhans cells were observed within and occasionally subjacent to the OGE. Scattered macrophages (Leu-M3 or OK Ia 1 positive) were observed in the inflammatory cell infiltrates and on the connective tissue papillae beneath the OGE. HLA-DR antigen reacting with OK Ia 1 was present on cells corresponding to OKT 6 positive cells in the OGE and subjacent to the OGE as well as in the inflammatory cell infiltrates beneath the PE and in the perivascular infiltrates. In some specimens HLA-DR antigen was also found to be associated with keratinocytes in the outer parts of the OGE. Occasional NK cells (Leu-7 positive) were localized inside and subjacent to the OGE. There was a considerable variation with respect to the number and distribution of the various mononuclear cells between specimens and from section to section from the same specimen.     

Extensive expression of TGF-b1 in chronically-inflamed periodontal tissue

The host immune response in chronic marginal periodontitis (CMP) raised against bacteria colonizing the dentogingival area is modulated by cytokines. This study examines the distribution of the transforming growth factor-beta1 containing (TGF-beta1+) cells in formalin-fixed and paraffin-embedded gingival specimens from 11 patients with chronic marginal periodontitis and 7 persons with healthy gingiva. Inflamed periodontal tissue contained a 100-fold more TGF-beta1+ cells than healthy gingiva. Diverse morphological TGF-beta1+ cell types were discerned. Double immuno-enzymatic and -fluorescence staining revealed that TGF-beta1+ cells comprised 21-29% macrophages 2-3% T-cells, 3-9% B-cells, 34-35% neutrophilic granulocytes and 7-10% mast cells. The densities of all TGF-beta1+ cell types in CMP were strongly increased in the connective tissue adjacent to the pocket epithelium, in the lamina propria and adjacent to the oral epithelium. In lesions with extensive inflammation, expression was also marked in pocket epithelium. TGF-beta1 is an immunosuppressive cytokine that stimulates wound healing. Upregulation of the cytokine in inflamed gingiva may counterbalance for destructive gingival inflammatory responses that are simultaneously taking place in patients with CMP.     

Juvenile and rapidly progressive periodontitis

Juvenile and rapidly progressive periodontitis are grouped under the heading of early-onset periodontitis. In recent years, much attention has been devoted to studying immunologic factors in early-onset periodontitis. This study was designed to investigate peripheral blood lymphocyte subpopulations, natural killer cells and interleukin-2 receptor positive (IL-2R +) cells in patients with juvenile and rapidly progressive periodontitis. 38 patients with juvenile and 30 patients with rapidly progressive periodontitis, plus 30 normal healthy control subjects were included in the study. Peripheral blood T-lymphocytes, helper T-cells, suppressor T-cells, HLA-DR+ cells, and IL-2R + cells were determined using appropriate monoclonal antibodies and the indirect immunofluorescence method. B-lymphocytes were identified using the direct immunofluorescence technique. Both groups of patients had normal number of total CD3+ T-cells, CD4+ helper T-cells, CD8+ suppressor T-cells, HLA-DR+ cells and IL-2R+ cells. Natural killer cells were found to be significantly elevated in both groups. These findings could contribute to the immunopathogenesis of early-onset periodontitis.     

牙周炎患者自身组织核酸刺激小鼠巨噬细胞后对破骨相关因子mRNA表达的影响

目的 检测牙周炎患者自身组织核酸刺激巨噬细胞后破骨相关因子白细胞介素-6（IL-6）、白细胞介素-12（IL-12）p35、IL-12p40、基质金属蛋白酶-9（MMP-9）、活化T细胞核因子 1（NFATc1）、核激活因子κB受体（RANK）、肿瘤坏死因子-α（TNF-α）mRNA的表达,观察牙周炎患者自身组织核酸对巨噬细胞向破骨细胞分化的影响作用。方法 采集翻瓣术中慢性牙周炎患者炎症牙周组织及正畸患者健康牙拔除术获取的健康牙周组织,提取组织总RNA逆转录cDNA。培养小鼠巨噬细胞系RAW264.7,加入质量浓度1 μg•mL-1的特定序列寡核苷酸MT01共孵育3 h后（以1 μg•mL-1的PBS作为对照）,加入已提取的炎症牙周组织及健康牙周组织cDNA（质量浓度为1 μg•mL-1）。实验分4组：健康组织cDNA,炎症组织cDNA,MT01+健康组织cDNA,MT01+炎症组织cDNA。4组细胞分别孵育3、6、12、24 h,采用实时定量聚合酶链反应法检测破骨相关因子IL-6、IL-12p35、IL-12p40、MMP-9、NFATc1、RANK及TNF-α mRNA的表达,进行两两组间比较。结果 牙周炎患者自身组织核酸可上调RAW264.7破骨相关因子IL-6、IL-12p35、IL-12p40、MMP-9、NFATc1、RANK及TNF-α mRNA的表达;在免疫抑制剂MT01的作用下,牙周炎患者自身组织核酸上调RAW264.7内破骨相关因子mRNA的表达状况受到抑制。结论 牙周炎患者自身组织核酸可以影响小鼠巨噬细胞向破骨细胞的分化。     

Flow-cytometric analysis of lymphocyte subsets in patients with advanced periodontitis

Peripheral blood lymphocytes from 36 patients with advanced periodontitis and from 34 healthy subjects were examined using a panel of monoclonal antibodies and fluorescence flow cytometry. The absolute and relative counts of B-cells, T-cells, T-helper (TH), T-suppressor/cytotoxic (TS) cells, activated T-cells and natural killer cells were assessed and the TH/S ratio was calculated. In the periodontitis patients, the TH/S ratio was 1.56 +/- 0.62 and in the controls 1.35 +/- 0.55. This was not statistically significant. A classification on the basis of sex also did not show significant differences in the TH/S ratio. B-cells and activated T-cells were slightly increased in the periodontitis group. However, all determined lymphocyte subpopulations revealed no significant differences between the groups. These results indicate that local inflammatory reactions and immunoregulatory dysfunction are limited to the periodontium in the patients with advanced periodontitis, with no significant quantitative effects on the peripheral lymphocyte subpopulations.     

Lymphocyte numbers and function in relation to periodontitis and smoking

BACKGROUND: T and B lymphocytes play important roles in periodontitis. Smoking is considered a risk factor for periodontitis and may exert its negative effects through leukocytes. Taking smoking into consideration, the aim of this study was to analyze numbers of circulating T (CD3+) cells and their CD4+ and CD8+ subpopulations, B (CD19+) cells, and T-cell proliferative capacity in periodontitis. METHODS: Lymphocyte immunophenotyping for T cells, their CD4+ and CD8+ subsets, and B cells was performed on peripheral blood from 76 periodontitis patients and 36 controls. Proliferative capacity of T cells was determined in whole-blood lymphocyte culture assays after mitogenic stimulation. RESULTS: Total T cells, CD4+ and CD8+ subpopulations, and responsiveness to specific T-cell stimuli did not differ between patients and controls; in addition, B cells were not significantly elevated in periodontitis patients. However, more periodontal breakdown in smoking patients was associated with higher numbers of CD3+ T cells, as well as with CD4+ and CD8+ T-cell subsets, and increased T-cell proliferation. Numbers of B cells were not affected by smoking. CONCLUSIONS: The increased numbers of T-cells and elevated T-cell responsiveness in patients who smoke may be one of several explanations why smoking is a risk factor for periodontitis. The mechanism of how T-cell function contributes to increase the severity of periodontal breakdown in smoking periodontitis patients needs to be investigated further.     

Expression of HLA class II antigens in marginal periodontitis of patients with Down's syndrome

The expression of HLA Class II antigens on the surfaces of immunocompetent cells and the presence of CDla⁺ cells (Langerhans cells) are important components of antigen presentation. Quantitative variations in HLA class II expression on antigen-presenting cells play a role in immune regulation. An indirect irnmunofluorescent technique was used on cryostat sections to reveal such differences qualitatively or quantitatively between chronic marginal periodontitis (CMP) in patients with Down's syndrome (DS) and in otherwise normal patients (NP). We found increased frequency of HLA Class II (HLA-expression on inflammatory cells and on keratinocytes of the oral gingival epithelium) in CMP of DS patients compared to sections from NP. The expression of HLA-DR was more frequent on the keratinocytes of the pocket epithelium in NP than in DS. There were significantly higher numbers of CD1a+ cells and ratios of HLA-DR+/CD1a⁺ cells and HLA-DP⁺/CD1a⁺ cells in the DS group compared to the NP group. Our conclusion is that there is a more pronounced inflammatory process in the gingival sites with CMP of DS patients compared to CMP in NP. The findings also indicate that there is a highly activated immune response in CMP of DS patients.     

In situ characterization of mononuclear cells in human dental periapical inflammatory lesions using monoclonal antibodies

Mononuclear cells in cryostat sections of human dental periapical inflammatory lesions were studied with the aid of murine monoclonal antibodies and with indirect immunofluorescence microscopy. T lymphocytes (OKT3-positive cells) made up a major part of the cells in the infiltrates. They were found mainly in clusters, although single cells were also seen. T helper cells (OKT4) were more numerous than suppressor/cytotoxic T cells (OKT8-positive cells), with a ratio of approximately 2:1. Langerhans cells (OKT6-positive cells) were not demonstrated: only a few scattered HNK 1-positive cells, probably natural killer cells, were detected. A large number of OKM1- and OKIa 1-positive cells were detected in the infiltrates. Their size and number varied considerably in the different areas of the sections. These cells are probably macrophages. Sheets of small OKIa 1-positive cells were also demonstrated, indicating the presence of B lymphocytes or activated T lymphocytes. The results indicate that immune reactions may be of importance in the pathogenesis of periapical inflammatory lesions.     

Characterization of mononuclear cells in cyclosporin A induced gingival enlargement

Abstract – Gingival biopsies from five patients with cyclosporin A (CsA) induced enlargement of the gingiva contained prominent mononuclear infiltrates beneath the epithelial basement membrane. These infiltrates and surrounding tissues were studied using monoclonal antibodies directed against lymphocytes and their subgroups (OKT 3, OKT 4, OKT 8, Leu 3 and Leu 4), monocytes (OKM 1) and the framework structure of HLA-DR antigens (OKI al). As revealed by the reactivity of these antibodies the vast majority of the mononuclear cells in two individuals consisted of monocytes and in the others of T-Iymphocytes. Virtually no B-lymphocytes were observed. The ratio between T-cells bearing T 4 and T 8 antigens, in separate biopsies, varied between 6 and 0.3. Control biopsies were obtained from the gingiva of seven healthy individuals without gingivitis. These biopsies contained relatively few mononuclear cells all of which had T-lymphocytc phenotype.     

Subpopulations of lymphocytes in connective tissue from phenytoin-induced gingival overgrowth

The presence of mononuclcar cells was studied in gingival biopsies from seven children exhibiting phenyloin(PHT)-induced gingival overgrowth, three children with gingivitis and a control group consisting of three children without clinical signs of inflammation. The mononucle-ar cells were detected using monoclonal antibodies defining functional T-lymphocytc subpopulations, B-lymphocytes and monocytes. Gingival biopsies from the individuals in the PHT-group showed a substantial number of mononuclear cells. The distribution of mononuclear cells in separate individuals were as follows: 69-95% OKT3 +/Leu4+ cells (T-lymphocytes), 50-64% OKT4 +/Leu3 + cells (T-helper phenotype) and 29-46% OKT8 + cells (T-suppressor/cytotoxic phenotype). None of the biopsies in the PHT-group contained more than a few scattered plasma cells. The vast majority of all mononuclear cells present in the biopsies reacted with OKIal, a monoclonal antibody defining the HLA-DR framework. In contrast, biopsies from the control group and the gingivitis group contained few mononuclear cells, the majority of which were T-cells. This suggests that immunologic reactions mediated by T-cells may play a role in the pathogenesis of the PHT-induced lesion.     

Subpopulations of lymphocytes in connective tissue from phenytoin-induced gingival overgrowth

The presence of mononuclear cells was studied in gingival biopsies from seven children exhibiting phenytoin(PHT)-induced gingival overgrowth, three children with gingivitis and a control group consisting of three children without clinical signs of inflammation. The mononuclear cells were detected using monoclonal antibodies defining functional T-lymphocyte subpopulations, B-lymphocytes and monocytes. Gingival biopsies from the individuals in the PHT-group showed a substantial number of mononuclear cells. The distribution of mononuclear cells in separate individuals were as follows: 69-95% OKT3 +/Leu4+ cells (T-lymphocytes), 50-64% OKT4 +/Leu3+ cells (T-helper phenotype) and 29-46% OKT8+ cells (T-suppressor/cytotoxic phenotype). None of the biopsies in the PHT-group contained more than a few scattered plasma cells. The vast majority of all mononuclear cells present in the biopsies reacted with OKIa1, a monoclonal antibody defining the HLA-DR framework. In contrast, biopsies from the control group and the gingivitis group contained few mononuclear cells, the majority of which were T-cells. This suggests that immunologic reactions mediated by T-cells may play a role in the pathogenesis of the PHT-induced lesion.     

Antigen-presenting cells in human periodontal disease tissues

T cells are present in the inflammatory infiltrates of periodontal disease lesions and require antigen presentation by antigen-presenting cells (APCs). While it is still not known whether Th1 or Th2 cells predominate in these lesions, it has been reported that different APCs may induce activation of different T-cell subsets. An immunoperoxidase technique was used to investigate the presence of CD1a+, CMRF-44+, CMRF-58+ and CD83+ dendritic cells, CD14+ macrophages or dendritic cell precursors and CD19+ B cells in gingival biopsies from 21 healthy or gingivitis and 25 periodontitis subjects. The samples were divided into three groups according to the size of infiltrate (group 1, small infiltrates; group 2, medium infiltrates; group 3, extensive infiltrates). The presence of numerous CD1a+ Langerhans cells was noted in the epithelium with no differences between the healthy/gingivitis and periodontitis groups. The percentage of CD83+ dendritic cells in the infiltrates was higher than the percentage of CD1a+, CMRF-44+ or CMRF-58+ dendritic cells. Endothelial cells positive for CD83 were found predominantly in areas adjacent to infiltrating cells, CD83+ dendritic cells being noted in the region of CD83+ endothelium. The percentage of CD14+ cells in the inflammatory infiltrates was similar to that of CD83+ dendritic cells. B cells were the predominant APC in group 2 and 3 tissues. The percentage of B cells in group 3 periodontitis lesions was increased in comparison with group 1 periodontitis tissues and also in comparison with group 3 healthy/gingivitis sections. Functional studies are required to determine the roles of different APC subpopulations in periodontal disease.     

Antigen‐presenting cells in human periodontal disease tissues

T cells are present in the inflammatory infiltrates of periodontal disease lesions and require antigen presentation by antigen‐presenting cells (APCs). While it is still not known whether Th1 or Th2 cells predominate in these lesions, it has been reported that different APCs may induce activation of different T‐cell subsets. An immunoperoxidase technique was used to investigate the presence of CD1a+, CMRF‐44+, CMRF‐58+ and CD83+ dendritic cells, CD14+ macrophages or dendritic cell precursors and CD19+ B cells in gingival biopsies from 21 healthy or gingivitis and 25 periodontitis subjects. The samples were divided into three groups according to the size of infiltrate (group 1, small infiltrates; group 2, medium infiltrates; group 3, extensive infiltrates). The presence of numerous CD1a+ Langerhans cells was noted in the epithelium with no differences between the healthy/gingivitis and periodontitis groups. The percentage of CD83+ dendritic cells in the infiltrates was higher than the percentage of CD1a+, CMRF‐44+ or CMRF‐58+ dendritic cells. Endothelial cells positive for CD83 were found predominantly in areas adjacent to infiltrating cells, CD83+ dendritic cells being noted in the region of CD83+ endothelium. The percentage of CD14+ cells in the inflammatory infiltrates was similar to that of CD83+ dendritic cells. B cells were the predominant APC in group 2 and 3 tissues. The percentage of B cells in group 3 periodontitis lesions was increased in comparison with group 1 periodontitis tissues and also in comparison with group 3 healthy/gingivitis sections. Functional studies are required to determine the roles of different APC subpopulations in periodontal disease.     

Characterization of mononuclear cells in cyclosporin A induced gingival enlargement

Gingival biopsies from five patients with cyclosporin A (CsA) induced enlargement of the gingiva contained prominent mononuclear infiltrates beneath the epithelial basement membrane. These infiltrates and surrounding tissues were studied using monoclonal antibodies directed against lymphocytes and their subgroups (OKT 3, OKT 4, OKT 8, Leu 3 and Leu 4), monocytes (OKM 1) and the framework structure of HLA-DR antigens (OKI al). As revealed by the reactivity of these antibodies the vast majority of the mononuclear cells in two individuals consisted of monocytes and in the others of T-lymphocytes. Virtually no B-lymphocytes were observed. The ratio between T-cells bearing T 4 and T 8 antigens, in separate biopsies, varied between 6 and 0.3. Control biopsies were obtained from the gingiva of seven healthy individuals without gingivitis. These biopsies contained relatively few mononuclear cells all of which had T-lymphocyte phenotype.     

Identification of inflammatory cell phenotypes in human oral carcinomas by means of monoclonal antibodies

Monoclonal antibodies reacting with human T cell sub-populations, Langerhans cells and macrophages were used to examine the quantitative distribution of immune-competent cells in normal oral mucosa and invasive oral carcinomas. Both immunofluorescent and immunoperoxidase procedures were applied. In normal oral epithelia, the dominant immune-reactive cell was the Langerhans cell, positive for OKT 6 and expressing HLA-DR gene products (OKIal⁺). Many intra-epithelial non-epithelial cells (non-keratinocytes), belonged to the lymphocyte system carrying the suppressor/cytotoxic phenotype (OKT 8⁺). This lymphocyte sub-population was also the most prominent cell type in the normal mucosal stroma. The quantitative evaluation of immune-competent cells in squamous cell carcinomas revealed elevated numbers of all the inflammatory cell sub-populations investigated (suppressor/cytotoxic lymphocytes, helper/inducer lymphocytes, Langerhans cells, macrophages) compared with the normal oral mucosa. There was a striking increase in suppressor/cytotoxic lymphocytes (OKT 8⁺) and in cells of the macrophage system, including Langerhans cells (OKIal⁺, OKM l⁺, OKT 6⁺). In the stroma distant to the tumour complexes, many helper/inducer lymphocytes (OKT 4⁺) were also observed.     

Identification of cells expressing T and p28,33 (la-like) antigens in sections of human chronic inflammatory periodontal disease

Previous studies have indicated that the lymphocytes present in progressive human chronic inflammatory periodontal disease may be of B-cell origin. The present investigation was undertaken to determine the usefulness of specific antisera to lymphocyte differentiation antigens in identifying the cell subpopulations in situ in the chronic inflammatory lesion. Both HuTLA⁺ (5–10%) and p28,33⁺ (90–95%) cells were identified. Although the p28,33⁺ population may include macrophages and a subset of T-cells, the majority of cells are probably of B-cell origin. This supports earlier suggestions that progressive periodontal disease in humans is a B-cell lesion and demonstrates that antisera to lymphocyte differentiation antigens in an immunofluorescence assay will delineate cell subpopulations in situ in a chronic inflammatory lesion.     

Interleukin 4 (IL-4) and IL-6-producing memory T-cells in peripheral blood and gingival tissues in periodontitis patients with high serum antibody titers to Porphyromonas gingivalis

We assessed cytokine production and proliferation of memory T-cells that were isolated from peripheral blood of adult periodontitis patients with high anti- Porphyromonas gingivalis titer. Memory T-cells were stimulated with P. gingivalis lipopolysaccharide, sonicates and formalin-killed whole cells. Interleukin 4 (IL-4)- and IL-6-producing cells were stained by immunocytochemically on peripheral blood smears and compared with cryostat sections of autologous gingival biopsies. Memory T-cells in the peripheral blood of patients rated significantly higher than in healthy subjects (32.3±7.1 vs 25,3±3.0%). Stimulation of patient-derived memory T-cells with P. gingivalis whole cells induced higher IL-4 production than in healthy subjects (4.4±4.1% vs 0.7±0.6%). Induction of IL-4-producing memory T-cells by P. gingivalis lipopolysaccharide and whole cells was respectively 1.37 and 1.56 times that induced by medium alone. IL-6 production did not differ between the groups. Proliferation of memory T-cells in healthy subjects tended to be more inhibited by P. gingivalis antigens than that in patients. In some patients, induction of IL-4- and IL-6-producing memory T-cells in peripheral blood and in autologous gingival biopsies tended to coincide. Memory T-cells with functional characteristics of Th2 could be a crucial cell population capable of reflecting individual susceptibility to periodontitis.     

Flow-cytometric analysis of lymphocyte subsets and mCD14 expression in patients with various periodontitis categories

BACKGROUND: Membrane-bound CD14 (mCD14) is expressed mainly on circulating monocytes and tissue macrophages. It is one of the receptors, which act at the recognition of lipopolysaccharides by host cells. Periodontopathic bacteria result in activation of cellular and humoral immune responses. AIM: The aim of the present study was to analyze the peripheral blood mCD14 concentrations as well as cell surface markers of lymphocyte subsets in periodontitis patients of various categories. MATERIALS AND METHODS: Peripheral blood samples were obtained from 22 early onset periodontitis (EOP), 10 adult periodontitis (AP) patients and 13 systemically and periodontally healthy control subjects. Three-color flow cytometry and a panel of relevant monoclonal antibodies were used to determine the percent expression of various cell surface markers on peripheral blood mononuclear cells (PBMCs). The results were tested statistically by one-way variance analysis and Newman Keuls test. RESULTS: No significant difference was observed between the study groups with regard to the relative counts of B-cells, T-cells, T-helper, T-cytotoxic/suppressor, activated T-cells and natural killer cells. EOP patients expressed significantly lower level of interleukin-2 receptor (IL-2R) when compared with AP patients (6.08% and 19.3% respectively) (p<0.05). The level of mCD14 in EOP patients (7.18%) was lower than that of AP patients (9.3%) and the control subjects (9.2%), but the differences were not statistically significant. CONCLUSIONS: The low level of IL-2R in the EOP group may be interpreted as an insufficient responsiveness to the periodontopathogens, which may be ultimately related with the more severe tissue destruction. Though not significant, the reduced expression of mCD14 in EOP group may also be related with the immune system deficiencies in these patients.     

Increased density of lymphocytes bearing gamma/delta T-cell receptors in recurrent aphthous ulceration (RAU)

Lymphocytes bearing the T-cell receptor (TCR) gamma/delta are increased in the peripheral blood of patients with recurrent aphthous ulcers (RAU) and Behcet's disease. In this study, we examined whether the density of TCR-gamma/delta bearing lymphocytes was also increased locally in RAU lesions. Ten RAU lesions from ten patients were compared with ulcer-free mucosa from sites contralateral to the lesions, and with 10 samples of clinically healthy oral mucosa taken from 10 healthy volunteers. Samples were labeled with a panel of monoclonal antibodies specific to CD3, alpha/beta TCR and gamma/delta TCR in avidin-biotin-peroxidase complex (ABC) staining. Lymphocytes expressing gamma/delta TCRs were very low in non-lesional mucosa and clinically healthy mucosa. By contrast, gamma/delta T-cells were numerous and observed in all RAU lesions especially within the epithelium, inflammatory infiltrates and at perivascular locations. The count of gamma/delta T-cells was high in connective tissue of RAU (200 +/- 126 cells/mm2) compared with connective tissue of controls (4+/-4 cells/mm2; P<0.0001) or non-lesional mucosa (5+/-7 cells/mm2). Interestingly, the density of gamma/delta T-cells was also high in the epithelium of RAU (70+/-34 cells/mm2) compared with the epithelium of non-lesional mucosa (2.8+/-06 cells/mm2; P<0.0001) or epithelium of healthy controls (1.2+/-1.5 cells/mm2; P<0.0001). Moreover, the mean percentage of gamma/delta+ T-cells among total CD3+ lymphocytes was increased in the connective tissue area from 4% and 5% in controls and non-lesional mucosa, respectively, to 19% in RAU. In epithelial areas, the average percentage was increased from 2% and 6% in controls and non-lesional mucosa, respectively, to 36% in RAU. These data showed that gamma/delta T-cells are more numerous in RAU lesions and such an increase was purely restricted to RAU inflammatory areas.