Heparanase expression in periapical granulomas and radicular cysts.

Heparanase is an endo-ß-D-glucuronidase enzyme which degrades heparan sulfate glycosaminoglycan side chains of proteoglycans in the extracellular matrix and in basement membranes. The aim of this study was to evaluate the expression of heparanase in periapical granulomas (PGs) and radicular cysts (RCs). Immunohistochemistry was used to assess heparanase expression in PGs and RCs. Parameters including stain intensity, location and cell type were used to characterize heparanase expression in the periapical lesions. Ordered categories (from weak to strong) were used to compare the level of heparanase staining in the PG and RC groups. Both epithelial cells and inflammatory cells were positive for heparanase. The relative staining of the epithelial cells was strong, whereas the relative staining of the inflammatory cells was weak. Significant differences in immunohistochemical staining of epithelial cells were observed between RCs and PGs (p = 0.002). The relative expression of heparanase in epithelial cells in RCs was strong. In PGs, lesions with few or no epithelial cells, heparanase was predominantly expressed weakly by inflammatory cells. PGs and RCs have the same infectious origin. Therefore, the different cellular sources of heparanase in these periapical lesions may imply that this enzyme has specific pathogenetic functions in RCs and PGs.

[Screening saliva].

Oral Fluids (OF) are a complex mixture including components deriving from, salivary glands, blood, nasal and bronchial secretions, mucosal lining cells and microbiota. Therefore, OF as a mirror of the body, were suggested as an important diagnostic fluid for the detection of both, oral and systemic diseases. OF as diagnostic fluids have several advantages; their collection is easy, inexpensive and noninvasive, they are suitable for home use and for epidemiology researches, they are easy to store and ship, do not clot and enable fast detection. OF based diagnostics research accomplished a great advance during the last decade. This is mainly due to biotechnology improvements such as 2-D Fluorescence Difference Gel Electrophoresis, quantitative Mass Spectrometry and bioinformatics systems. These technologies enabled the detection of more than 3000 proteins in oral fluids, as well as the establishment of a panel of biomarkers to different human pathological conditions (i.e. periodontitis, Sjögren's Syndrome, oral cancer, pancreatic cancer etc). However, this diagnostic field has several drawbacks, mainly due to oral fluids variance composition, blood contamination as a result of gingivitis or mucosal injuries, the lack of a single established collection protocol and the presence of high abundant components in OF. This article summarizes the current research, and provides an outlook toward the foundation of this unique body fluid as a major player in the diagnostic field.

Sialochemistry and cortisol levels in patients with Sjogren's syndrome.

OBJECTIVES: (i) To determine whether salivary cortisol and electrolyte levels differ between patients with Sjogren's syndrome (SjS) and healthy individuals. (ii) To assess correlations between whole-saliva cortisol and some clinical manifestations in patients with SjS. METHODS: A total of 24 healthy women (mean age 49.3±9.8) served as controls (C) vis-à-vis 17 patients with SjS (mean age 55.5±15.7). Salivary cortisol concentration was determined, and sialochemistry analysis was performed. RESULTS: Significantly lower saliva flow rates and higher salivary chloride (Cl(-) ), potassium (K(+) ), and Ca(2+) levels were found in the SjS group. No significant differences or correlations were found in other parameters, including sodium (Na(+) ), magnesium (Mg(2+) ), phosphate ((-) ), urea (U), and salivary cortisol levels. CONCLUSION: Increased whole-salivary output of Cl(-) and K(+) in SjS may reflect release from apoptotic rests of acinar cells after secondary necrosis. Normal levels of salivary Na(+) , Mg(2+) , and (-) argue against concentration effect, deranged tubular function or cortisol (mineralocorticosteroid) effect as the cause for these findings. Increased salivary Ca(2+) levels probably reflect leakage of plasma Ca(2+) through the injured oral mucosa in SjS. In spite of disease-associated stress, salivary cortisol, a stress biomarker, was not increased, suggesting insufficient hypothalamus-pituitary-adrenal (HPA) axis response and/or local consumption of cortisol by lymphocyte infiltrates.

Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG.

OBJECTIVES: The aim of this study was to examine whether triple depletion of salivary-α-amylase (sAA), albumin (Alb) and immunoglobulins G (IgGs) may improve the visualization capability of proteins in two-dimensional gel electrophoresis (2-DE) of oral fluids (OF). SUBJECTS AND METHODS: Oral fluids from healthy volunteers were subjected sequentially to sAA removing device followed by application to an Alb and IgG immunoaffinity column (triple depletion). The depleted OF samples were analyzed using SDS-PAGE followed by 2-DE and protein identification using ion-trap mass spectrometry (MS). RESULTS: This specific triple depletion technique unmasked spots never visualized before. A total of 36 new spots were observed after depletion (348 vs 312 before depletion). Moreover, 58 spots showed more than twofold increase intensity after depletion. In the 60-69kDa area, the depletion procedure unmasked 14 proteins including HSP70, LTA4H, L-Plastin, Desmoplakin that are known to be involved in disease pathogenesis. CONCLUSION: The ability to selectively remove and elute the most abundant OF proteins visualized on the 2-DE represents an important step in the characterization of human OF. The better visualization and gel resolution achieved will improve quantification abilities in 2-DE and in tag-MS leading to better identification of disease-specific biomarkers. We further analyzed the eluted Alb and IgGs isoforms suggesting a new methodology venue for OF.

Comparative proteomic analysis of human oral fluids according to gender and age.

BACKGROUND: Recently, interest in finding disease bio-markers in human body fluids including oral fluids (OF), mainly saliva has increased. However, the physiologic differences in salivary proteins according to gender and age should be explored to establish a clinical diagnostic tool. OBJECTIVE: To compare OF protein expression according to gender and age, using proteomic approaches. MATERIALS AND METHODS: Oral fluids from 27 healthy volunteers (14 males, 13 females) was collected and divided into three age-groups. OF proteins were separated by means of 2D-SDS-PAGE. A total of 51 proteins in 37 protein spots were identified by ESI-MS/MS. RESULTS: Gender differences revealed six proteins with significant higher expression in females, including ß-2-microglobulin and transferrin. Age differences revealed decrease in expression of eight proteins with aging among males and seven proteins differentially expressed with aging among females including prolactin inducible protein, Ig-k light chain, transferrin, and calgranulin-B. CONCLUSION: Proteomic analysis of OF revealed differences in protein expression according to gender and age and therefore can highlight future use of this technique for diagnostic purposes in health and in disease.

Possible proteomic biomarkers for the detection of pancreatic cancer in oral fluids.

The 80% mortality rate of pancreatic-cancer (PC) makes early diagnosis a challenge. Oral fluids (OF) may be considered the ultimate body fluid for non-invasive examinations. We have developed techniques to improve visualization of minor OF proteins thereby overcoming major barriers to using OF as a diagnostic fluid. The aim of this study was to establish a short discriminative panel of OF biomarkers for the detection of PC. Unstimulated OF were collected from PC patients and controls (n = 30). High-abundance-proteins were depleted and the remaining proteins were analyzed by two-dimensional-gel-electrophoresis and quantitative dimethylation-liquid-chromatography-tandem mass-spectrometry. Label-free quantitative-mass-spectrometry analysis (qMS) was performed on 20 individual samples (n = 20). More than 100 biomarker candidates were identified in OF samples, and 21 had a highly differential expression profile. qMS analysis yielded a ROC-plot AUC value of 0.91 with 90.0% sensitivity and specificity for a combination of five biomarker candidates. We found a combination of five biomarkers for PC. Most of these proteins are known to be related to PC or other gastric cancers, but have never been detected in OF. This study demonstrates the importance of novel OF depletion methodologies for increased protein visibility and highlights the clinical applicability of OF as a diagnostic fluid.

Different proteomic protein patterns in saliva of Sjögren's syndrome patients.

OBJECTIVE: To investigate the salivary protein profile in patients with Sjögren's syndrome (SS), and healthy control subjects. MATERIALS AND METHODS: Unstimulated whole saliva samples were collected from 16 age-matched females; eight healthy subjects and eight patients diagnosed with SS (six primary SS, one incomplete SS and one primary SS associated with B cell lymphoma). Proteins were extracted and separated individually by 2D sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Selected protein spots of interest were analysed by electrospray ionization--tandem mass spectrometry. Obtained data were searched against the Swiss-Prot and NCBI non-redundant protein databases using Mascot software. RESULTS: Two groups of patterns of protein expression were observed in the eight SS patients: a major group (six patients) with significant expression differences from the healthy subjects and the second group (two patients) with a pattern similar to the eight healthy subjects. CONCLUSION: In this preliminary study, protein expression differences were found between SS patients and healthy subjects. Individual analysis of SS patients exhibited two patterns of protein expression with no direct relation to the clinical, serological or histological severity of disease. This study emphasizes the difficulty of the present proteomic knowledge to diagnose and monitor the sequel of SS development.

Cortisol daily rhythm in saliva of healthy school children.

AIM: To investigate the daily rhythm of cortisol levels in saliva of school children. SUBJECTS AND METHODS: Probands (10-14 years, both genders) were recruited via personal contact and school visits. Exclusion criteria included hormonal and dental treatments during the trial, pharmaceuticals containing cortisol, or poor oral hygiene. Each volunteer collected 20 saliva samples during one day at defined times starting immediately after waking up and ending at night. Additionally, they completed a sampling diary. Saliva samples were analysed in duplicate using a commercial cortisol luminescence kit. RESULTS: Cortisol concentration in saliva followed a daily rhythm. Within 20 minutes after waking up cortisol reached the highest level of 9.69 (+/-3.89) nmol/L. After 90 minutes cortisol concentration decreased linearly by 50% and stagnated at 4.14 (+/-1.93) nmol/L for 3 to 8 hours. Thereafter, levels decreased gradually reaching almost zero after 14 hours. Overall, no gender-specific differences in saliva cortisol levels were observed except for 3 time points: 3, 10 and 11 hours after waking. CONCLUSION: This study establishes guidelines for a normal secretion pattern, plus explores pain level measurements and their correlation to saliva cortisol levels in this age group.

Proteomic analysis of protein components in periodontal ligament fibroblasts.

BACKGROUND: Characterization of periodontal ligament (PDL) fibroblast proteome is an important tool for understanding PDL physiology and regulation and for identifying disease-related protein markers. PDL fibroblast protein expression has been studied using immunological methods, although limited to previously identified proteins for which specific antibodies are available. METHODS: We applied proteomic analysis coupled with mass spectrometry and database knowledge to human PDL fibroblasts. RESULTS: We detected 900 spots and identified 117 protein spots originating in 74 different genes. In addition to scaffold cytoskeletal proteins, e.g., actin, tubulin, and vimentin, we identified proteins implicated with cellular motility and membrane trafficking, chaparonine, stress and folding proteins, metabolic enzymes, proteins associated with detoxification and membrane activity, biodegradative metabolism, translation and transduction, extracellular proteins, and cell cycle regulation proteins. CONCLUSIONS: Most of these identified proteins are closely related to the extensive PDL fibroblasts' functions and homeostasis. Our PDL fibroblast proteome map can serve as a reference map for future clinical studies as well as basic research.

The enamelin (tuftelin) gene.

This paper reviews the primary structure, characteristics and possible function of tuftelin/enamelin protein. It describes the distribution of tuftelin in the ameloblast cell and in the extracellular enamel matrix, employing high resolution protein-A gold immunocytochemistry. The chromosomal localization of the human tuftelin gene and its possible involvement in autosomally linked Amelogenesis Imperfecta, the most common hereditary disease of enamel, is also discussed.

Expression of the gene for the receptor of gonadotropin-releasing hormone in the rat mammary gland.

Recent findings have demonstrated that the GnRH gene is expressed in the mammary gland of pregnant and lactating rats but not of virgin rats. Indeed, significant concentrations of biologically active GnRH have been found in milk of human, cow, sheep and rat. We have, therefore, looked for expression of the GnRH receptor in the rat mammary gland. By reverse transcription (RT)-PCR amplification, we have demonstrated the presence of GnRH receptor mRNA in mammary gland samples derived from virgin, pregnant and lactating rats. The GnRH receptor transcript cloned from the mammary gland was sequenced and found to have an identical coding region to the one cloned from the pituitary gland. In addition, we have found that the mammary gland, as the pituitary gland, contains at least two transcripts having the same coding region but different 5' non-coding regions. Binding studies, however, could demonstrate only low-affinity binding sites. These results, therefore, suggest that the regulation of the GnRH receptor occurs posttranscriptionally rather than at the level of transcription.

Thymic humoral factor, THF-gamma 2, enhances immunotherapy of murine cytomegalovirus (MCMV) infection by both CD4+ and CD8+ immune T cells.

Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. The contribution of the different spleen cell subsets in conferring curative immunocytotherapy to fatally MCMV-infected immunosuppressed mice was assessed using adoptive immunotherapy. It was found that the efficacy of passively transferred immune spleen cells is dose dependent and that the therapeutic effect can be enhanced considerably by treating donor mice with thymic humoral factor (THF-gamma 2). Polymerase chain reaction (PCR) of the donor spleen population was negative, indicating that no MCMV-DNA was transferred with the immune cells. Analysis of the donor mice after THF-gamma 2 treatment showed increased levels of CMV-neutralizing antibodies, while enhancement of natural killer (NK) activity was transient and lasted only during the early phase of the infection. FACS analysis demonstrated that treatment with THF-gamma 2 restored the size of both cell subsets CD4+ and CD8+ that were decreased following MCMV infection. It is shown that both CD4+ and CD8+ T-cell subsets participate in controlling the development of the fatal disease in MCMV-infected immunosuppressed recipients. It is suggested that the enhancement of the immunocompetence of both populations of spleen cells from treated donors is mediated in part by the restoration of Interleukin-2 (IL-2) production by THF-gamma 2.

Treatment of murine cytomegalovirus salivary-gland infection by combined therapy with ganciclovir and thymic humoral factor gamma 2.

An optimal therapeutic regimen against primary CMV salivary-gland infection has not yet been developed. We used a murine CMV (MCMV) model system to assess the ability of combined thymic humoral factor THF-gamma 2 immunotherapy and ganciclovir (GCV) antiviral chemotherapy to eliminate detectable viral DNA from salivary glands of infected animals. Mice in different experimental groups were inoculated intraperitoneally with MCMV, treated, and then sacrificed either 2 weeks or 3 months later. To amplify and detect MCMV DNA in infected salivary-gland tissue, we developed a sensitive polymerase chain reaction (PCR) using a glycoprotein B gene primer pair that amplifies a 356 bp segment. During the acute phase of the infection, the detection of high titers of infectious virus in the salivary glands correlated with a strong PCR amplification signal. Although active virions could not be recovered from untreated animals 3 months after viral inoculation, the PCR assay detected a latent MCMV genome. Treatment with either GCV alone or THF-gamma 2 alone had little or no effect on the presence of MCMV DNA. By contrast, combined treatment with THF-gamma 2 and GCV significantly reduced the amount of salivary-gland MCMV DNA to below the limit of PCR detection. The results presented here, and experimental data from previous MCMV research in our laboratories, imply that elimination of the virus from the salivary glands could be due in part to THF-gamma 2 restoration of the various MCMV-suppressed, cell mediated immune-responses. Combining THF-gamma 2 immunotherapy and GCV antiviral chemotherapy may be an important step toward an effective therapeutic regimen that has the potential to prevent the establishment of viral latency ensuing from primary MCMV salivary-gland infection.

Characterization of murine autologous salivary gland graft cells: a model for use with an artificial salivary gland.

The purpose of this study was to examine the growth and key functional abilities of primary cultures of salivary epithelial cells toward developing an artificial salivary gland. Cultures of epithelial cells originating from submandibular glands of BALB/c mice were established. Parenchymal cells were isolated by a Percoll gradient technique and thereafter seeded on irradiated NIH 3T3 fibroblasts serving as a feeder layer. The isolated cells were termed autologous salivary gland epithelial (ASGE) cells and could be cultivated for at least five passages (time limit of experiments). ASGE cells presented the typical organizational behavior of epithelial cells and electron microscopy, as well as immunostaining for cytokeratins, confirmed their epithelial origin. Furthermore, measurements of transepithelial resistance and water permeability indicated the ability of the ASGE cells to form a functional epithelial barrier. This study suggests that primary salivary epithelial cells can be obtained that exhibit critical characteristics needed for use with an artificial secretory device.

Development of a highly sensitive quantitative competitive PCR assay for the detection of murine cytomegalovirus DNA.

Viral persistence and molecular latency are characteristic of infection by the human cytomegalovirus (HCMV). Using the murine cytomegalovirus (MCMV) as a model for human infection, a quantitative-competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify MCMV-DNA in the salivary glands of infected mice. The QC-PCR detected high numbers of MCMV DNA copies in the absence of infectious virus. By comparing the DNA content and the results obtained from a standard semiquantitative plaque assay, it is concluded that 1 plaque-forming unit (pfu) is the equivalent of approximately 1500 viral genomes. By day 42-post infection (pi) 4x10(3) copies of DNA/1 mg tissue were sufficient to reactivate infectious virions after cyclophosphamide immunosupression. By day 90 pi, however, when the DNA load was decreased to <1.2x10(2), reactivation was not observed. These results indicate that viral reactivation will occur when the number of infectious DNA copies is equivalent about 2-3 pfu. This quantitative test may therefore help to detect CMV and the risk of reactivation in immunosupressed patients.

Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing.

The abundant hydrophobic, proline-glutamine, and histidine-rich (over 90%) amelogenins constitute the major class of proteins in forming extracellular enamel matrix. These are thought to play a major role in the structural organization and mineralization of developing enamel. The present report describes the successful sequencing of the major human amelogenin protein, by use of both Edman degradation and cDNA sequencing. When Edman degradation was used, over 75% of the primary structure of the protein was determined. This sequence was supplemented with cDNA sequencing studies, which revealed the predicted sequence of this protein. Together, they provide the complete sequence of an important human enamel protein. The information complements recent studies on bovine and human amelogenin genes. A comparison between the present results and the protein sequences predicted from the corresponding human amelogenin genomic coding regions and that of cDNA sequences of other species is described.

Interferon-gamma deficiency attenuates local P. gingivalis-induced inflammation.

Infection with the periodontal pathogen Porphyromonas gingivalis causes a strong local inflammatory reaction. Using IFNgamma-deficient mice, we tested the hypothesis that the absence of IFNgamma would result in a reduction of the local pro-inflammatory response to P. gingivalis. Cytokine secretion by macrophages from IFNgamma(-/-) animals was significantly attenuated. Addition of IFNgamma restored cytokine secretion. In vivo injection of P. gingivalis into subcutaneous chambers increased the intra-chamber leukocyte counts and TNFalpha and IL-1beta levels. This increase was significantly lower in the IFNgamma(-/-) mice. Local reconstitution of IFNgamma(-/-) mice at the site of inflammation with the IFNgamma gene increased the levels of TNFalpha and decreased the IL-10 levels. Anti-P. gingivalis IgG1 levels, a marker of Th2 response, were higher in immunized IFNgamma(-/-) than in IFNgamma(+/+) mice. The results suggest that lack of IFNgamma reduced the amplitude of the local pro-inflammatory response without decreasing the humoral protective response. The higher IgG1/IgG2a ratio observed supports the possibility of a Th2-dominant response in IFNgamma-deficient animals.

The effect of mechanical force on mRNA levels of collagenase, collagen type I, and tissue inhibitors of metalloproteinases in gingivae of dogs.

Orthodontic force causes an injury to and subsequent degradation of the attachment apparatus, thus leading to the transposition of the tooth. The gingiva, however, is compressed and sometimes becomes hypertrophic with tooth movement and often shrinks after treatment. To study the effect of force on the gingiva, we applied orthodontic force in dogs and analyzed gingival tissues 1, 2, 3, 7, 14, and 28 days later as well as after removing the force. The effect of force on mRNA levels of collagen type I (col-I), matrix metalloproteinase-1 (MMP- 1), and tissue inhibitors 1 and 2 (TIMPs) genes was analyzed by RT-PCR, and MMP-1 activity was determined by zymography. The results showed that force significantly increased both the mRNA levels of MMP-1 and its interstitial activity. After the removal of force, MMP-1 gene expression was significantly decreased. The results could partly explain the clinically observed shrinkage and adaptation of the gingiva during tooth movement.

Inverse dose- and time-dependent effect of basic fibroblast growth factor on the gene expression of collagen type I and matrix metalloproteinase-1 by periodontal ligament cells in culture.

BACKGROUND: Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament cells (PLC). The aim of this study was to assess the dose-dependent effect of bFGF administration on the levels of gene expression of collagen type I (a1) (col I), collagen type III (col III), and collagenase-1 (MMP-1) in PLC. METHODS: PLC were cultured in different concentrations of bFGF (0.1 to 10 ng of bFGF) for 14 and 21 days. At each time point, the gene expression of the examined molecules was assessed semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULTS: The results indicated that bFGF exhibits an inverse time- and dose-dependent effect on the gene expression of col I and MMP-1: it simultaneously downregulates the gene expression of col I and upregulates the gene expression of MMP-1. On the other hand, bFGF had no dose-dependent effect on col III gene expression. The effect of bFGF on the expression of the three genes was modulated by the time of incubation with bFGF. CONCLUSIONS: These results suggest that bFGF is one of the important regulators involved in the active remodeling of col I in the periodontal ligament and possibly in other connective tissues.

The effect of centrifugal force on the transcription levels of collagen type I and collagenase in cultured canine gingival fibroblasts.

An orthodontically treated tooth is often destabilized in its newly corrected location and relapses towards its original position. Hitherto, the explanation for this phenomenon was that orthodontic force brings about "stretching" of gingival collagen fiber, which "pull back" the tooth towards its pretreatment position. A previous ultrastructural study showed that after force application the gingival collagen fibres were torn, laterally spaced and of increased diameter. Therefore, they could not "pull back" the tooth and be the cause of the relapse. In the present study, in order to find a more plausible explanation at the molecular level, the effect of pressure on the gene transcription of collagen type I and tissue collagenase was examined by semiquantitative, reverse transcriptase-polymerase chain reaction assay. Attached buccal gingiva was excised from anaesthetized dogs and gingival fibroblasts were grown in culture. Following application of pressure (0.167 kg/l g cell mass), the transcription of collagen type I was increased while that of tissue collagenase was decreased. These results corroborate the ultrastructural in vivo findings that orthodontic force is associated with larger amounts of collagen type I in the gingiva.