Immunohistochemical investigation of lysozyme, lactoferrin, α1-antitrypsin, α1-antichymotrypsin and ferritin in parotid gland tumors

Presence of lysozyme, lactoferrin, α₁-antitrypsin, α₁-antichymotrypsin and ferritin was examined by the immunoperoxidase method in 15 consecutive parotid gland tumors as well as in normal parotid gland tissue. Lysozyme and lactoferrin were detected in intercalated duct cells of normal tissue and in the epithelial component of pleomorphic adenomas. α₁-antitrypsin, α₁-antichymotrypsin and ferritin were found in both epithelial and mesenchymal components of pleomorphic adenomas but not in normal parotid tissue. In the epithelial components of adenolymphoma only α₁-antichymotrypsin and lactoferrin were observed. The results would support a tentative histogenic link between the intercalated duct cell and the epithelial component of the pleomorphic adenoma.     

Changes in the expression of αv integrins in oral squamous cell carcinomas

Integrins are cell surface adhesion molecules that regulate normal cellular interactions; aberrant integrin expression is believed to play a role in tumour invasion and metastasis. The α_v subunit is capable of forming heterodimers with several β subunits but not all the heterodimers expressed in oral epithelium have been investigated. We have examined the distribution of α_v integrins in normal buccal mucosa and seventeen oral squamous cell carcinomas. Antibodies to the α_v subunit and α_vβ₅ heterodimer stained normal epithelium, with strong expression in the basal layers and weaker staining in the more superficial layers. The β₃ and β₆ subunits were not expressed in normal epithelium. Anti-α_v and anti-α_vβ₅ antibodies stained all the squamous cell carcinomas, but the pattern of expression was variable both within and between tumours. Poorly differentiated tumours showed the weakest staining and often had areas showing loss of expression. β₆, was expressed in all of the carcinomas, indicating new expression of the α_vβ₆ integrin in malignant oral epithelium. These results suggest that alterations in α_v integrins may contribute to the behaviour of malignant epithelium and that α_vβ₆ expression may play a role in tumour progression.     

Integrin receptors and their relationship to cellular proliferation and differentiation of oral squamous cell carcinoma. A quantitative immunohistochemical study

The expression of extracellular matrix (ECM) proteins (fibronectin, laminin, collagen IV) and ECM receptors of integrin type (α₂β₁, collagen receptor; α₆ chain of the fibronectin receptor; α₆ chain of the laminin receptor) were examined in normal oral squamous epithelium and in invasive areas of oral squamous cell carcinomas with various differentiation and proliferation activities (Ki-67 antigen labelling), evaluating the presence, quantity (using an image analysis system) and distribution of the integrin subunits. In the mucosa, there was uniform immunostaining for α₂β₁ and α₆ concentrated at the cell membrane in the basal/supra basal cell zone, whereas, α₅ showed a discontinuous staining of the basal cell-basement membrane interface. α₂ and α₆ could be visualized in all carcinomas α₅ showed low expression preferentally in less differentiated carcinomas. In contrast to normal mucosa, there was an increase in α₆ staining in well-differentiated carcinomas. Dedifferentiation of oral carcinomas was accompanied by an increase in cellular proliferation and with a decrease in α₂β₁ and α₆ staining. This reduction of α₆ staining was shown to be statistically significant, suggesting that this integrin may be a valuable grading parameter for oral squamous cell carcinoma.     

α1-Proteinase inhibitor in gingival crevicular fluid of humans with adult periodontitis: serpinolytic inhibition by doxycycline

The serum protein, α₁-proteinase inhibitor (α₁PI), defends the host against serine proteinases, e.g. PMN elastase. Using a rabbit anti-serum against human α₁-PI, this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot. GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaCl₂ buffer, pH 7.6. α₁-PI concentration increased with G and was highest in AP subjects. H sites only showed intact α₁-PI (52 kDa); no degradation fragments (48 kDa) were detected. In G and AP subjects, α₁-PI degradation fragments were seen in 17% and 71% of GCF samples, respectively. Both collagenase and α₁-Pl-degrading activities in GCF increased with severity of inflammation (GCF flow). Moreover, the α₁-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline. We propose: (1) that collagenase promotes periodontal breakdown not only by degrading collagen, but also by depleting α₁-PI regulation of elastase and other serine-proteinases, thereby favoring a broader attack on extracellular matrix (ECM) constituents, and (2) based on a recent longitudinal double-blind study using the techniques described above for α₁-PI analysis, that low-dose doxycycline administration to humans with adult periodontitis can inhibit this broad cascade of ECM degradation.     

Creep and β1 formation in dental amalgam

abstract — Measurements of creep, β₁ phase content and γ ₁ lattice parameter were performed on four conventional amalgams stored at 37°C up to 2 years. The creep values and lattice parameters decreased and the β₁, content increased during this period; the most prominent alterations took place within the first 6 months.     

The potential role of α2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis

Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma α₂-macroglobulin (α₂M). Both 50- and 95 kDa gingipain R were efficiently inhibited by α₂M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, α₁-inhibitor-3 a-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the aminio acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of α₂M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by α₂M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.     

Electron microprobe analysis of the γ2 phase in dental amalgam

A bstract — The microstructure and composition of the γ₂ phase in four conventional lathe-cut or spherical amalgams were investigated using an electron microprobe.
In addition to its presence in the matrix, γ₂ phase was found, in lathe-cut amalgams, in contact with ε (Cu₃Sn) phase, separated from γ (Ag-Sn) phase by a thin band of γ₁ phase and, rarely, in direct contact with γ phase, and, in spherical amalgams, within the relict of partly reacted spherical particles. Inclusions of γ, γ ₁ and Cu-Sn phase were occasionally identified within regions of γ₂ phase. The major element composition of the γ₂ phase was similar in all amalgams (83.0 to 83.2 per cent Sn, 17.0 to 16.8 per cent Hg).     

Cathepsin B, α2-macroglobulin and cystatin levels in gingival crevicular fluid from chronic periodontitis patients

Abstract. Gingival crevicular fluid (GCF) was collected from 16 molar and premolar sites in each of 20 chronic periodontitis patients before and after periodontal therapy using filter paper strips. These were eluted individually into buffer for determination of cathepsin B and its endogenous inhibitors, α₂-macroglobulin and cystatin. Cathepsin B activity was assayed with a fluorogenic peptide substrate, α₂-macroglobulin by enzyme-linked immunosorbent assay and cystatin activity by inhibition of papain. Total amounts of enzyme and inhibitor per GCF sample decreased after treatment and correlated positively with pocket depth and gingival, bleeding and plaque indices. These comparisons were nearly always statistically significant for pooled site data and sometimes so for mean patient values. The amounts of α₂-macroglobulin and cystatin were greater than those of cathepsin B and, surprisingly, enzyme and inhibitor levels correlated positively with each other. Experiments with purified reagents, however, demonstrated that the cathepsin B: α₂-macroglobulin complex was still active against the low molecular weight substrate and that cystatin levels in GCF are probably insufficient to inhibit the enzyme substantially. These factors may explain why GCF cathepsin B activity reflects the clinical status of periodontal lesions and has been identified in another study as a promising indicator of disease progression.     

Serum β2 microglobulin in oral malignancy and premalignancy

Scrum β₂ microglobulin concentrations measured by radioimmunoassay in patients with oral squamous cell carcinoma, or with oral keratosis and epithelial dysplasia, were found to be significantly increased compared with age and sex matched controls.     

Stoichiometric and X-ray diffraction analysis on the γ2→η' transformation in a dispresant phase silver amalgam

Abstract— Phase composition of an amalgam prepared from a two-particle alloy was determined over a 2-year period by X-ray diffraction. The γ₂content decreased from 3.6% to 0.3%, and η' increased from 3.9% to 10/0%. These alterations in ph content agreed with stoichiometric calculations performed on the basis of the solid state reaction: γ₂+Ag/Cu→η'+γ₁     

Histological observations of hard tissue barrier formation in amputated dental pulp capped with α-tricalcium phosphate containing calcium hydroxide

Abstract This study was performed to evaluate the pulpal response to α-tricalcium phosphate (αTCP) containing calcium hydroxide (Ca(OH)₂). The dental pulps of monkeys were amputated and dressed with four agents: αTCP, αTCP containing 1% Ca(OH)₂, αTCP containing 5% Ca(OH)₂, and Ca(OH)₂ being used as a control. The pulpal responses were histologically evaluated after 4 and 8 weeks. The pulp tissue treated with αTCP proliferated above the level of the original wound surface, and a thin layer of hard tissue barrier was formed directly against the capping agent. The barrier demonstrated atubular matrix lined with flattened or cuboidal cells, but occasionally appeared irregular in form. Ca(OH)₂ dressing resulted in destruction of pulp tissue, with a thick hard tissue barrier being formed below the level of the exposure site. The barrier consisted coronally of osteodentin and pulpally of tubular dentin lined with odontoblast-like cells. By contrast, 1% Ca(OH)₂ added to αTCP produced a slight proliferation of pulp tissue. An atubular matrix barrier, pulpally lined with cuboidal cells, formed above the exposure site. It was later followed by the formation of tubular matrix lined with columnar cells. Teeth treated with 5% Ca(OH)₂, showed a thin necrotic layer and a thick barrier formation. The barrier was composed of tubular dentin-like tissue lined with odontoblast-like cells. It would appear that αTCP containing a small amount of Ca(OH)₂ may be clinically useful as a capping agent, as it induced consistent hard tissue formation, without excessive destruction of underlying pulp tissue.     

Periodontal-derived cells attach to cementum attachment protein via α5β1 integrin

A specific collagenous cementum attachment protein (CAP) has been identified in human cementum which promotes selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro. The CAP is known to support attachment of periodontal-derived cell via an RGD motif, which suggests an integrin-mediated mode of attachment. The purpose of the present study was to ascertain which integrin(s) are involved in the attachment of periodontal-derived cells to CAP. The integrins examined comprised subunits of the major receptors for fibronectin (alpha 5) and collagen (alpha 2, alpha 3), as well as the common beta 1 subunit which is present in many extracellular matrix receptors. The wells of 48-well non-tissue culture treated plates were coated with CAP (2 micrograms/ml). For negative and positive controls the wells were coated with bovine serum albumin and fibronectin (5 micrograms/ml), respectively. Human gingival fibroblasts and periodontal ligament fibroblasts were labeled with [3H]-proline, incubated with anti-integrin antibodies and added to the precoated wells. Attachment was assessed after incubating the cells for 1 h at 37 degrees C in the presence of the antibodies. Antibodies to alpha 5 and beta 1 inhibited the attachment of both human gingival fibroblasts and human periodontal ligament fibroblasts to CAP, while anti alpha 2 and alpha 3 antibodies did not affect the attachment. The binding of the fibroblasts to fibronectin was also inhibited by anti-alpha 5 and beta 1 antibodies, both of which are components of the "classical" fibronectin receptor and remained unaffected by the addition of anti-alpha 2 and alpha 3 antibodies. Proteins migrating in SDS-polyacrylamide gels in positions similar to the alpha 5 and beta 1 integrin subunits were present in fractions bound to a column of CAP coupled to Sepharose CL-4B. These results indicate that the attachment to CAP of the periodontal-derived cells, human gingival fibroblasts and human periodontal ligament fibroblasts, is mediated primarily via the integrin alpha 5 beta 1.     

Corrosion of non –γ2– amalgams

Abstract – Abstract– Eight different dental amalgams were studied in an in vitro corrosion test. Cylindrical test specimens were stored in weak lactic acid solutions at different pH for 1 month. The concentrations of mercury and copper in the solutions were measured and corrosion depths estimated. The results indicated that more copper was released from non - γ₂ - amalgams compared with conventional ones. The amounts of copper from these amalgams were not directly correlated to the concentration in the alloys. Mercury was released in much lower amounts than copper for all amalgams and seemed to originate from the γ₁-phase. A low pH promoted the corrosion.     

Immunohistochemical study of lymphoid tissue in human fetal salivary gland

Immunoreactivity of lysozyme (LY), lactoferrin (LF), α₁ antichymotrypsin (α₁-ACT), α-antitrypsin (α₁-AT), keratin proteins KL1, PKK1, K8.12, S-100 protein, MAM-3, MAM-6, and epithelial membrane antigen (EMA) were evaluated in lymphoid and glandular tissues of developing salivary gland of human fetus (gestational age ranging from 17 to 40 wk to investigate the role of lymphoid tissue in developing salivary glands. In a total of 79 cases, lymphoid cell aggregations were noted in parotid (57 cases), submandibular (21 cases) and sublingual (5 cases) glands. Mononuclcar cells showing intense activity of LY, α₁-ACT and α₁-AT were present in the lymphoid aggregation. The glandular ducts embedded in lymphoid tissue were negative to MAM-3, MAM-6, EMA and S-100 protein, but showed positive PKK1 and KL1 reaction during early stages of development, and showed degeneration and effacement upon increase in number and LY activity of the mononuclear cells. The lymphoid aggregations progressively emerged as lymph nodes.     

A possible association of α1-antitrypsin deficiency with the periodontal condition in adults

Abstract. α₁-antitrypsin (α1AT) is a serum inhibitor of several neutrophil-derived proteolytic enzymes, the serine proteinases. In certain individuals, a deficiency of this enzyme inhibitor is present, whereby only 10 to 20% of the normal concentration is present in the circulation. The purpose of this study was to evaluate the influence of this α1AT deficiency on the periodontal condition in a case control study 10 patients aged 20–50 years with a mean age of 32 and showing a deficiency of αlAT were randomly selected from a national data bank, which contains persons known to be affected with the αlAT deficiency state. Control subjects were matched for age, gender and socio-economic status. Full periodontal charting on all subjects was performed on 6 sites per tooth, including probing attachment level, probing depth, bleeding on probing and a plaque score. Analysis of the data revealed no differences with regard to attachment loss between the two groups. The deficiency group showed 9.3% pockets ≥5 mm and the matched control group 5.9%. When these results were covariated with plaque scores, the deficiency group appeared to have a higher number of sites with probing depths ≥5 mm.     

Effects on salivary flow rate and composition of withdrawal of and re-exposure to the β1 -selective antagonist metoprolol in a hypertensive patient population

Secretion rates and composition of unstimulated and chewing–stimulated whole saliva and 3% citric acid stimulated parotid and submandibular-sublingual secretions were studied in 12 hypertensive patients during withdrawal of and re-exposure to antihypertensive pharmacotherapy. All the patients' blood pressures were well controlled by monotherapy with metoprolol, a β₁-selective adrenoceptor antagonist. Blood pressure measurements and saliva sampling were performed at about 9.30 a.m., 2 h after intake of breakfast, on days 0 (medicated baseline), 7, 14, 28 (nonmedicated experimental values and nonmedicated baseline) and 35 (medicated experimental values). A significant increase in unstimulated whole saliva secretion rate was observed when metoprolol was withdrawn and a corresponding decrease when the drug was reintroduced. A positive correlation was found between diastolic blood pressure levels and chewing-stimulated whole saliva secretion rates. In unstimulated whole saliva and 3% citric acid stimulated submandibular-sublingual secretion, the output of total protein, amylase, potassium, calcium and phosphate was significantly increased during the withdrawal period and decreased when metoprolol was reintroduced. For chewing-stimulated whole saliva, the corresponding changes were restricted to output of total protein and amylase while for citric acid stimulated parotid secretion, no changes in salivary composition were observed. Finally, in all secretions one or both of the ratios hexosamine/total protein and sialic acid/total protein were affected, indicating a possible effect of (J-adrenoceptor antagonists on salivary protein synthesis.     

Levels of prostaglandin E2, thromboxane, and prostacyclin in periodontal tissues

Prostaglandin E₂ (PGE₂), thromboxane A₂ (TXA₂), and prostacyclin (PGI₂) are naturally occurring metabolites of arachidonic acid which have been associated with inflammation. To assess the relative importance of these mediators in periodontal diseases, the levels of all three were measured by radioimmunoassay in human tissues removed during surgery. TXA₂ and PGI₂ were determined as their stable hydrolysis products thromboxane B₂ (TXB₂) and 6-keto-prostaglandin F_1α (6-K-PGF_1α), respectively. Tissues from periodontal pockets were separated into superficial (n = 8) and deep (n = 22) samples. Noninflamed gingival samples (n = 3) were taken from distal wedges with no clinical evidence of periodontal disease. Most of the samples taken from deep sites (73%) had measurable PGE₂ with a mean level of 122 pg/mg. Half of these samples also had measurable TXB₂ which correlated positively with levels of PGE₂. Half of the superficial gingival samples had detectable levels of PGE₂, and none had detectable TXB₂. 6-K-PGF_1α was found in virtually all samples but at somewhat lower levels in noninflamed tissue. Neither PGE₂ nor TXB₂ were detected in noninflamed samples.     

Increased bonding of a glass-ionomer cement to dentin by means of FeC13

Abstract – It was previously demonstrated by other authors that FeC1₃ is readily sorbed by dentin surfaces. The main purpose of the present work was to investigate the possibility of increasing the bonding of a glass-ionomer cement to dentin by means of FeC1₃. It was found that the strength of the bond was approximately doubled when the dentin was treated with an acidic cleansing agent followed by an application of an aqueous solution of FeC1₃ of suitable concentration.     

Decrease of γ1 lattice constant with time in dental silver amalgam

Abstract— As a measure of the mercury content of the γ₁ phase in dental silver amalgam, the lattice constant of this phase in different amalgams was determined at various times after trituration. The lattice constants of amalgams prepared from alloys with a high silver content have lower values and show a steeper decline with time than amalgams with a low Ag-content. Therefore, since a higher lattice constant presumably is associated with increased mercury vapor, the mercury vapor emission from amalgams with a low Ag-content probably is greater than from amalgams with a high Ag-content, especially during the first weeks after trituration.     

TNF-α release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS

Lipopolysaccharide (LPS), a cell wall component of Gram negative anaerobic bacteria, has been implicated in the pathogenesis of periapical disease resulting from infected root canals. Calcium hydroxide [Ca(OH)₂] has been shown to be an effective medicament in such infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)₂ may also be the result of direct inactivation of LPS. The aim of this study was to investigate whether the toxic potential of an Escherichia coli LPS could be reduced or eliminated by Ca(OH)₂. Four concentrations of E. coli LPS ranging from 1–1000 ng/ml sterile water were incubated in duplicate either with 25 mg Ca(OH)₂ or sterile water alone. Controls consisted of Ca(OH)₂ without LPS or sterile water only. Monocytes were collected from peripheral blood by centrifuging through a gradient and plated to a specific density. Adherent monocytes were incubated for 4 days at 37°C with 5% CO₂ in M199 medium with 10% autologous serum. The different LPS solutions were added to the wells on day 5. After 4 h the supernatants were collected and quantitatively assayed for TNF-α using a commercial ELISA kit. Statistical analysis was performed with ANOVA . Results indicated that Ca(OH)₂ is able to eliminate the ability of an E. coli LPS to stimulate TNF-α production in peripheral blood monocytes ( P <0.0001).