The effect of age on proliferating cell nuclear antigen expression in oral gingival epithelium of healthy and inflamed human gingiva

BACKGROUND: Age-related changes in proliferative activity in human gingival epithelium are uncertain. Proliferating cell nuclear antigen (PCNA) is a nuclear protein associated with the cell cycle. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. The aims of this study were to investigate the localization of PCNA expression in oral gingival epithelium (OGE) and to define the age-related changes as to PCNA-proliferative index (PI) in inflamed as well as healthy gingiva. Mitotic index (MI) was also used as a conventional marker of cell proliferation. Additionally, the effect of aging upon the maximum epithelial thickness (MET) was determined. METHODS: Twenty older (65 to 85 years) (study) and 20 middle-aged (35 to 45 years) (controls) subjects were included in the study. Biopsies were obtained both from healthy and inflamed gingiva. The expression of PCNA was evaluated in formalin-fixed, paraffin-embedded gingival samples using an immunoperoxidase technique and PC 10 monoclonal antibody to PCNA. Hematoxylin and eosin stained sections were used for the quantitative measurement of MI and MET. RESULTS: All the tissue sections contained positive staining cells for PCNA in the gingival epithelium. Although PCNA expression was observed both in the basal and suprabasal layers, it was more prominent in the suprabasal layers. PI in inflamed gingiva was significantly higher in the older group. However, no significant difference was observed between the study and control groups with respect to PI in healthy gingiva. When all the subjects taken into the study were analyzed as a single group, PI in the inflamed gingival samples were found to be increased with aging. Nevertheless, no age-related change was noted in MI and MET. In both the study and the control groups, PI, MI, and MET were found to be increased due to inflammation. CONCLUSIONS: Our data indicate that PCNA expression in inflamed gingiva is higher in older subjects. Furthermore, a significant correlation was noted between aging and PCNA expression in inflamed gingiva. As there is no increase in mucosal epithelial thickness despite increased proliferation, we speculate that the duration of the PCNA+ phase in cell cycle may be longer in older subjects. This study also implies that PCNA immunolocalization can be used as an index of the state of cell proliferation in both biological and pathological events of the gingiva and/or other mucosal tissues.     

纤维调节素在大鼠和人类牙龈组织中的差异性表达

目的:研究纤维调节素在大鼠和人类健康和牙龈组织中的分布与表达。方法:用免疫组织化学的方法研究纤维调节素在大鼠牙龈及人类健康和炎性牙龈组织中的分布。与之比较,I型胶原纤维在鼠磨牙牙龈组织中的分布也做了研究。结果:在大鼠牙龈中,纤维调节素分布在牙龈上皮基底上层,在腭侧牙龈结缔组织有强表达。I型胶原纤维也有类似的分布。比较而言,纤维调节素在颊侧牙龈结缔组织表达较弱。对于人类,纤维调节素在炎性牙龈结缔组织中表达明显,然而在健康牙龈结缔组织中表达较弱。结论:与Ⅰ型胶原纤维关系密切的纤维调节素在颊侧和腭侧牙龈中有差异性表达,并且在人类炎性牙龈组织中表达增强。     

The effect of smoking on epithelial proliferation in healthy and periodontally diseased marginal gingival epithelium

BACKGROUND: Smoking causes an increase in the thickness of gingival epithelium, which is the outcome of increased keratinocyte proliferation or loss. Smoking-related changes in the proliferative activity of the gingival epithelium are largely uncharacterized for periodontal diseases. The aim of the present study was to determine the effects of smoking on the proliferation of the epithelium in periodontally diseased marginal gingiva by comparing the expression patterns of two different proliferation markers. METHODS: Gingival biopsies (N=60) were obtained from smokers who had clinically healthy gingiva (n=10), smokers with gingivitis (n=10), smokers with periodontitis (n=10), non-smokers with clinically healthy gingiva (n=10), non-smokers with gingivitis (n=10), and non-smokers with periodontitis (n=10). The quantitative measurement of maximum epithelial thickness was performed on hematoxylin and eosin-stained sections. The expression patterns for proliferating cell nuclear antigen (PCNA) and Ki67 were evaluated immunohistochemically. RESULTS: The percentage of PCNA-positive cells was higher than the percentage of Ki67-positive cells in all groups (P<0.001). When the mean values of PCNA and Ki67 were compared in each group, a statistically significant difference was observed only in the healthy smoker group (P=0.003). Significant differences in PCNA proliferation indices were only found between the smoker group and the non-smoker healthy group (P=0.015). CONCLUSIONS: Smoking had an affect on the proliferation of cells in the oral gingival epithelium, regardless of periodontal status. The increase in thickness of the epithelium was not associated with smoking; periodontal status and inflammation seemed to be more important factors. Smoking induced the replication activity of gingival epithelium and induced DNA repair.     

The Expression of Adhesion Molecules and Proliferative Activity in the Human Gingival Epithelium

The aims of this study were to investigate the proliferative activity and the localization of adhesion molecules in human healthy and inflamed portions of gingiva. Gingival biopsies were taken under informed consent from 33 patients undergoing gingivectomy at the Dental Hospital of Iwate Medical University. Adhesion molecules such as E-cadherin (of adherens junction-related molecules), desmoglein (of desmosomal cadherins), integrin α6 and integrin, β4 (of hemidesmosome-related molecules) were studied immunohistochemically to analyze the patterns of expression in the gingival epithelia. Desmoglein was detected intercellularly in the spinous layer of the lining epithelia, but not in the junctional epithelium (JE). Integrin α6 was confined to the basement membranes facing the connective tissue of all parts of the gingival epithelia. Integrin β4 was found in the basal layer of the epithelia, and it was markedly found in all layers of the JE. The expression patterns of adhesion molecules in the pocket epithelium (PE) were similar to those in the JE. The proliferative activity was analyzed by the bromodeoxyuridine (BrdU) labeling method and immunohistochemical staining for Ki-67. BrdU-positive cells were found in the basal layer of the sulcular (SE) and oral epithelia (OE), but not in the JE, and were particularly localized in the suprabasal layer at the bottom of the SE. Meanwhile, proliferative activity was found in the PE with the progression of ueriodontitis.     

A histochemical study of glycosidases in hydantoin induced hyperplastic, healthy and inflamed human gingiva

Fresh samples of hydantoin induced hyperplastic, healthy and inflamed human gingiva were studied histochemically using various azo dyes for β-glucuronidase (EC 3.2.1.31), for β- and β-glucosidase (EC 3.2.1.20 and 3.2.1.21) as well as β-galactosidase (EC 3.2.1.23) and for N-acetylglucosaminidase (EC 3.2.1.30), in order to add support to the hypothesis that hydantoin induced hyperplasia is always connected with inflammation.
Moderate β-glucuronidase activity was observed in healthy, inflamed and hydantoinhyperplastic gingiva. The distribution of the enzyme activity was similar in all types of the tissue except the stratum corneum. The healthy gingiva did not reveal this activity whereas the inflamed and hydantoinhyperplastic gingiva did. The stratum basale and spinosum of the epithelium, the fibroblasts and the inflammatory cells, especially the macrophages, revealed enzyme activity in all types of tissues. In the healthy tissue only a few inflammatory cells were seen and thus the β-glucuronidase activity was low when compared to inflamed or hyperplastic gingiva.
Weak β-galactosidase, N-asetylglucosaminidase and β-glucosidase activity was seen in all types of gingival samples. Enzyme activity was observed in the same structures as β-glucuronidase with the exception of the stratum corneum, which revealed no activity.
The relatively strong β-glucuronidase activity in the keratinized cell layer of the epithelium of inflamed and hydantoinhyperplastic tissue may be due to the microbial enzyme diffusion into the keratinized cell layer of the injured tissue.     

Localization of interleukin-1 (IL-1) mRNA-expressing macrophages in human inflamed gingiva and lL-1 activity in gingival crevicular fluid

The exact cell type and site(s) involved in interleukin-1 (lL-1) production during gingival inflammation was determined by combining immunohistochemistry and in situ hybridization. IL-1 messenger RNA (mRNA)-expressing cells in human inflamed gingiva were identified as macrophages. The rate of IL-α mRNA expression in these macrophages was the same as IL-1 β mRNA expression. The rate of IL-1 mRNA expression was higher in connective tissue furthest from the pocket epithelium, although more macrophages were present at the connective tissue subjacent to the pocket epithelium. The IL-1 activity in gingival crevicular fluid (GCF) obtained from inflamed gingiva was higher than that from healthy gingiva and decreased after periodontal therapy. The IL-1 activity in GCF was almost completely abolished by the addition of anti-IL-1α antibody but not by anti-IL-1 β antibody, indicating that IL-1α is the predominant form in GCF. However, the IL-1 activity in GCF was unrelated to the number of IL-1 mRNA-exprerssing macrophages in the same gingival site where the GCF was obtained at the same time. The results suggest that macrophages in the connective tissue subjacent to the oral epithelium contribute to the production of IL-1 but those in connective tissue subjacent to the pocket epithelium play a different role in the generation of gingival inflammation.     

Human gingival collagenase in periodontal disease: the release of collagenase and the breakdown of endogenous collagen in gingival explants

Collagen degradation in different stages of gingival inflammation was examined. Higher collagenolytic activity and nonspecific protease activity were found in the severely inflamed gingiva, as compared to mildly inflamed or healthy gingiva. This was shown by electrophoretic studies and measurement of peptide-bound hydroxyproline in the culture.     

Hemoglobin concentration and oxygen saturation of clinically healthy and inflamed gingiva in human subjects

The hemoglobin concentration (Hb index) and oxygen saturation (apparent SO2) in human gingiva were estimated by tissue reflectance spectrophotometry (TRS). The gingiva had significantly lower Hb index and higher apparent SO2 than those in alveolar mucosa, but there was no difference in either parameter among different gingival areas. The reproducibility in repeated measurements was high for both Hb index and apparent SO2 in gingiva. In inflamed gingiva, Hb index was significantly higher than that in clinically healthy gingiva. A lower apparent SO2 was observed in inflamed gingiva. This suggests that the increase in blood supply is insufficient to meet the oxygen demand in inflamed gingiva. There were significant correlations between either the Hb index or the apparent SO2 and the clinical parameters of gingival inflammation such as gingival index, plaque index, Periotron score and probing depth. Thus, TRS may be clinically available to estimate the blood volume and oxygen saturation in inflamed gingiva.     

A histochemical study of arylaminopeptidases in hydantoin induced hyperplastic, healthy and inflamed human gingiva

Arylaminopeptidase activity in hydantoin induced hyperplastic, inflamed and healthy human gingiva was studied using various N-L-aminoacyl-2-naphthylamines as substrates. The activity was seen to be located in the basal cell layer of the epithelium in the entire connective tissue, but it was strongest just below the epithelium in all tissues. High enzymic activity was also observed in the inflammatory cells as well as in the capillary walls of hydantoinhyperplastic and inflamed gingiva. Strong enzymic activity was obtained when N-L-alanyl-, N-L-methionyl- and N-L-leacyl-2-naphthylamine were used as substrates. Moderate activity was observed with N-L-arginyl-2-naphthylamine and N-Llysyl-2-naphthylamine in other tissues except in healthy gingiva where the enzymic activity was low or nil. To test the possible involvement of aminopeptidase B in the material the reactions were performed both in the presence and absence of 0.2 M sodium chloride, which specifically activates this enzyme. There was no observable enzymic activity in any slices when N-L-prolyl-2-naphthylamine was used as substrate.
The role of different arylaminopeptidases in connection with gingival hyperplasia caused by diphenylhydantoin and the evident absence of enzymes specific to N-L-prolyl-2-naphthylamine are discussed.     

Localization of the neuropeptide galanin in nerve fibers and epithelial keratinocytes of the rat molar gingiva

Knowledge of the histochemical substrates of cellular and neurovascular connections in the gingiva is essential in order to understand the initial mechanisms of inflammation in the periodontium. Since the localization of the neuroendocrine peptide galanin in the gingiva is still unclear, we used immunohistochemical, in situ hybridization and immunoblot techniques to assess the localization of galanin in the gingiva of rat molars. Galanin-immunoreactive nerve fibers were located around blood vessels in the lamina propria, beneath the epithelium, in the epithelial-proprial junction and in the basal layer of the epithelium. Galanin was highly expressed in the suprabasal keratinocytes of the gingival epithelium. The localization of galanin in gingival nerve fibers and the expression of galanin in keratinocytes of the gingival epithelium indicate that galanin may be a possible regulator of different cellular functions in the gingiva.     

Histochemical study of labial salivary glands in Sj?gren's syndrome.

Human labial salivary gland biopsies of patients presenting connective tissue diseases associated with Sj?gren's syndrome were submitted to a polysaccharide histochemistry study. The normal acinar secretion is an association of neutral polysaccharides with a sulphosialomucin. In Sj?gren's syndrome, there is a great reduction in the secretory activity of the acinar cells, but no qualitative change was observed. The pathogenesis of this decreased production and its importance regarding the clinical manifestations of Sj?gren's syndrome are discussed.     

Immunohistochemical distribution of keratin proteins in clinically healthy human gingival epithelia

In clinically healthy/subclinically inflamed biopsies of marginal gingiva, the immunohistochemical distribution of keratin proteins was studied in junctional (JE), sulcular (SE), oral gingival (OGE) and in a few samples of alveolar mucosal epithelium (AE) by means of various mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. All regions stained in a nearly similar way with AE3 (keratins 1-8, all cells) and BE14 (keratin 5, basal and supra/parabasal cells). AE8-staining (keratin 13, supra/parabasal and spinous cells) was primarily confined to the stratified, nonkeratinized epithelia SE and AE, but also a variable part of JE and less frequently OGE were positive. The parakeratinized OGE was distinct in showing a homogeneous staining with AE2 (keratins 1/2, 10) and AE5 (keratin 3) throughout spinous cell layers. These antibodies did not stain JE and AE whereas SE stained in a scattered way with AE5 and sometimes also with AE2. The latter finding might indicate initial keratinization at molecular level. The JE was distinct in retaining basal characteristics throughout the epithelium with PKK2 (keratin 7, 16, 17, 19) and BE14 (keratin 5) although some initial suprabasal maturation, as observed with AE8, cannot be excluded. Differences in keratin staining of gingival epithelia and the AE was found with respect to AE1-reactivity (keratins 10, 14-16, 19) which was suprabasal in JE, SE and OGE but basal in AE.     

Immunohistochemical distribution of keratin proteins in clinically healthy human gingival epithelia

Abstract – In clinically healthy/subclinically inflamed biopsies of marginal gingiva, the immunohistochemical distribution of keratin proteins was studied in junctional (JE), sulcular (SE), oral gingival (OGE) and in a few samples of alveolar mucosal epithelium (AE) by means of various mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. All regions stained in a nearly similar way with AE3 (keratins 1–8, all cells) and BE 14 (keratin 5, basal and supra/parabasal cells). AE8-staining (keratin 13, supra/parabasal and spinous cells) was primarily confined to the stratified, nonkeratinized epithelia SE and AE, but also a variable part of JE and less frequently OGE were positive. The parakeratinized OGE was distinct in showing a homogeneous staining with AE2 (keratins 1/2, 10) and AE5 (keratin 3) throughout spinous cell layers. These antibodies did not stain JE and AE whereas SE stained in a scattered way with AE5 and sometimes also with AE2. The latter finding might indicate initial keratinization at molecular level. The JE was distinct in retaining basal characteristics throughout the epithelium with PKK2 (keratin 7, 16, 17, 19) and BE14 (keratin 5) although some initial suprabasal maturation, as observed with AE8, cannot be excluded. Differences in keratin staining of gingival epithelia and the AE was found with respect to AE 1 -reactivity (keratins 10, 14–16, 19) which was suprabasal in JE, SE and OGE but basal in AE.     

Cytokine concentrations in stimulated whole saliva among patients with primary Sjögren's syndrome, secondary Sjögren's syndrome, and patients with primary Sjögren's syndrome receiving varying doses of interferon for symptomatic treatment of the condition: a preliminary study

Sj?gren's syndrome is an autoimmune disorder which causes diminished salivary flow due to autoimmune sialoadenitis. This decrease in saliva flow is the result of inflammation and atrophy of the salivary glands. Most treatment regimens are palliative in nature, but treatment with interferon (IFN) holds promise for Sj?gren's syndrome sufferers. Several studies have investigated cytokine concentrations in the salivary glandular tissues from Sj?gren's syndrome patients; however, there is little information concerning cytokine expression in saliva. This is especially true with respect to treatment modalities and their effects on local cytokines. A clinical study was conducted to determine salivary interleukin (IL)-6, IFN, and IL-2, concentrations among subjects diagnosed with primary and secondary Sj?gren's syndrome and a healthy control group. The primary Sj?gren's syndrome showed significantly higher salivary IL-2 and salivary IL-6 than the control and secondary Sj?gren's groups. There were no between group differences for salivary IFN concentrations. In addition, the study assessed salivary IL-6, IFN, and IL-2 concentrations among 18 Sj?gren's syndrome patients before and after administration of IFN via the oral mucosal route. The results of the study showed that the mean values for the pre- and post-treatment groups for stimulated whole saliva flow rates were 3.15 and 3.74 ml/5 min, respectively. The post-treatment group exhibited a 16.8% increase in stimulated whole saliva flow rates. The salivary IL-6 concentration was 53.3% lower for the post-treatment group (17.79) as compared to the baseline value (33.35). The values for salivary IFN and salivary total protein were virtually unchanged from their baseline values. Salivary IL-2 values, however, were 50% lower in the post-treatment group (3.07) when compared to their respective baseline values (6.10). The results of this study suggest that healthy individuals exhibit lower salivary IL-2 and IL-6 as compared to individuals suffering from primary and secondary Sj?gren's syndrome. The results also suggest that administration of IFN via the oral mucosal route may increase salivary flow rates and depress certain cytokines (IL-2, IL-6) associated with inflammatory destruction of salivary glandular tissues in Sj?gren's syndrome patients.     

Complement factors in gingival crevice material from healthy and inflamed gingiva in humans

The presence and levels of complement factors C3, C4, C5, and C3 proactivator, were determined by electroimmuno assay in gingival cervice material from five individuals with healthy gingiva and from six patients with chronically inflamed gingiva. Higher concentrations of C3 and C4 were found in samples from chronically inflamed gingiva when compared to those healthy gingiva. The amount of C3 in material from healthy as well as from inflamed gingiva was related to that in plasma, when the albumin in plasma and gingival crevice material was used as a reference. The same was found for C4 in samples from healthy gingiva but in those from inflamed gingiva, the values were significantly lower when related to plasma levels. C5 could not be found in material from healthy gingiva but was present in material from inflamed gingiva. C3 proactivator was present in material from inflamed gingiva in the converted form. No C3 proactivator was found in material from healthy gingiva with the methods used. Analysis of C3 in samples from inflamed gingiva, using crossed immunoelectrophoresis, showed that C3 was converted in these samples. The results indicate that the complement system may be activated in gingival crevice material from inflamed gingiva.     

Localization and expression pattern of amelotin,odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.     

Identification of Langerhans cells in human gingival epithelium

The purpose of this study was to qualitatively compare three recent techniques of Langerhans cells detection in oral epithelium and to quantitatively compare Langerhans cells in clinically normal and clinically inflamed human gingival biopsies. Eleven subjects were selected who displayed chronic periodontitis and moderate gingival inflammation. A quadrant associated with clinically inflamed tissues was not treated, while the remaining teeth were scaled and root-planed. Two gingival biopsies were taken: clinically normal, treated tissue; and clinically inflamed, untreated tissue. Langerhans cells were stained using HLD-DR, S-100 and OKT6. They were quantitated using a standard grid for OKT6-stained sections only. Approximately 5 times as many Langerhans cells were identified in the biopsy specimens of clinically inflamed human gingiva as in clinically normal gingiva of the same patient. Of the methods studied, OKT6 was qualitatively determined to be the best for visualization of these cells. An immunologic role in the host response to chronic periodontal disease is postulated for Langerhans cells.     

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.     

Demonstration of tissue collagenase activity in vivo and its relationship to inflammation severity in human gingiva

To directly demonstrate both the presence and in vivo activity of tissue collagenase (EC 3.4.24.7) during gingival inflammation, tissue extracts from 54 specimens of variously inflamed human gingiva were analyzed individually for: a) collagenase-specific collagen degradation products, and b) collagen-bound collagenase. The TC^A collagen degradation product, identified using SDS-PAGE, was shown in 13/19 (68.4%) of insoluble tissue-residue fractions extracted from moderate-to-severely inflamed gingiva, but in only 2/21 (9.6%) slight-to-mildly inflamed gingival specimens, suggesting differences in the in vivo collagenase activity between the two groups. Using in vitro collagenase assays, gingival collagenase (as bound to insoluble collagen) was demonstrated in 92.8% of the moderate-to-severely inflamed gingival specimens and in 50% of the slight-to-mildly inflamed gingival specimens. A relationship was established between the active and latent forms of the enzyme and the degree of inflammation. Active enzyme was present in 78% of the moderate-to-severely inflamed specimens and in 14% of the slight-to-mildly inflamed gingiva. In contrast, latent collagenase was predominant in the slight-to-mildly inflamed group (86% of coliagenase-positive samples) compared with 46% of coliagenase-positive moderate-to-severely inflamed gingival samples. The collagen-bound gingival collagenase was inhibited by metal ion chelators, sulphydryl reagents and 10% FBS, but not by serine nor thiol-proteinase inhibitors and is therefore a neutral metalloproteinase.