Association between the baseline gene expression profile in periapical granuloma and periapical wound healing after surgical endodontic treatment.

In this study, we have investigated the association between the baseline gene expression profile in periapical granuloma and periapical wound healing after surgical endodontic treatment. Twenty-seven patients aged between 15 and 57 years underwent periapical surgery. The retrieved periapical tissue sample was used for mRNA expression analysis of COL1A1, VTN, ITGA5, IL-4, TNF, ANGPT, VEGFA, and CTGF. All patients were recalled after 6 and 12 months for periapical healing evaluation. Healing was then correlated with baseline gene expression. Healing was observed in 15 patients at the end of 6 months, which increased to 21 patients after 12 months. Six patients showed no healing even after 12 months. Analysis of baseline expression levels of the tested genes with healing status showed the mean relative expression of VTN, VEGFA, ANGPT, TNF, and CTGF to be significantly different (p < 0.05) between the healing group (6 and 12 months) (72.99%) and the non-healing (94.42%) group. Periapical Index scores 3-5 exhibited a positive correlation with ITGA-5 expression. Overexpression of ANGPT and a strong positive correlation between ITGA5 and PAI scores in the non-healing group of patients may suggest these genes to be a potential prognostic biomarker for periapical wound non-healing after surgical endodontic treatment.

Role of S100A4 in the Pathogenesis of Human Periapical Granulomas.

BACKGROUND/AIM: S100A4 expression is associated with the pathology of chronic inflammatory diseases. In this study, we investigated the role of S100A4 and four inflammatory mediators (IL-1ß, IκB, IL-10, and TNF-α) in human periapical granulomas (PGs). MATERIALS AND METHODS: S100A4 expression in PGs obtained by apicoectomy was examined by immunohistochemistry. Further, the expression of S100A4 and four inflammatory mediators was compared between PGs and healthy gingival tissues (HGTs) using real-time PCR. RESULTS: In the PGs, S100A4 was found to be expressed in endothelial cells and fibroblasts. Furthermore, real-time PCR revealed that the expression of S100A4 and IL-1ß in PGs was significantly higher than that in HGTs. Although a correlation between the expression of S100A4 and IκB or IL-10 was not detected, a positive correlation between the expression of S100A4 and IL-1ß or TNF-α was observed. CONCLUSION: The expression of S100A4 correlates with the pathogenesis of PGs.

Transforming growth factor beta-1 expression in macrophages of human chronic periapical diseases.

The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-ß1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-ß-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm2) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-ß1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-ß1 expressing macrophages varied with human chronic periapical diseases. The TGF-ß1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

MMP1-1607 polymorphism increases the risk for periapical lesion development through the upregulation MMP-1 expression in association with pro-inflammatory milieu elements.

OBJECTIVE: In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. MATERIAL AND METHODS: MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p<0.05 was considered statistically significant). RESULTS: The MMP1-1607 1G/2G and 1G/2G+2G/2G genotypes were significantly more prevalent in the patients than in controls, comprising a risk factor for periapical lesions development. MMP-1 mRNA levels were higher in periapical lesions than in healthy periodontal ligament samples, as well as higher in active than in inactive lesions. The polymorphic allele 2G carriers presented a significantly higher MMP-1 mRNA expression when compared with the 1G/1G genotype group. The ordered logistic regression demonstrated a significant correlation between the genetic polymorphism and the expression levels of MMP-1. Additionally, the pro- and anti-inflammatory cytokines IL-17A, IFN-g, TNF-a, IL-21, IL-10, IL-9, and IL-4 were significant as complementary explanatory variables of MMP-1 expression. CONCLUSION: The MMP1-1607 SNP was identified as a risk factor for periapical lesions development, possibly due to its association with increased MMP-1 mRNA levels in periapical lesions. The MMP-1 expression is also under the control of the inflammatory milieu elements, being the cytokines TNF-a, IL-21, IL-17A, and IFN-g associated with increased MMP-1 levels in periapical lesions, while IL-10, IL-9, or IL-4 presented an inverse association.

MMP1-1607 polymorphism increases the risk for periapical lesion development through the upregulation MMP-1 expression in association with pro-inflammatory milieu elements

ABSTRACT Increased matrix metalloproteinases (MMPs) activity is a hallmark of periapical granulomas. However, the factors underlying the MMPs expression modulation in healthy and diseased periapical tissues remains to be determined. Objective In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. Material and Methods MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p<0.05 was considered statistically significant). Results The MMP1-1607 1G/2G and 1G/2G+2G/2G genotypes were significantly more prevalent in the patients than in controls, comprising a risk factor for periapical lesions development. MMP-1 mRNA levels were higher in periapical lesions than in healthy periodontal ligament samples, as well as higher in active than in inactive lesions. The polymorphic allele 2G carriers presented a significantly higher MMP-1 mRNA expression when compared with the 1G/1G genotype group. The ordered logistic regression demonstrated a significant correlation between the genetic polymorphism and the expression levels of MMP-1. Additionally, the pro- and anti-inflammatory cytokines IL-17A, IFN-g, TNF-a, IL-21, IL-10, IL-9, and IL-4 were significant as complementary explanatory variables of MMP-1 expression. Conclusion The MMP1-1607 SNP was identified as a risk factor for periapical lesions development, possibly due to its association with increased MMP-1 mRNA levels in periapical lesions. The MMP-1 expression is also under the control of the inflammatory milieu elements, being the cytokines TNF-a, IL-21, IL-17A, and IFN-g associated with increased MMP-1 levels in periapical lesions, while IL-10, IL-9, or IL-4 presented an inverse association.

Gene-expression analysis of matrix metalloproteinases 1 and 2 and their tissue inhibitors in chronic periapical inflammatory lesions.

BACKGROUND: Periapical inflammatory lesions have been investigated previously, but understanding of pathogenesis of these lesions (granulomas and radicular cysts) at the molecular level is still questionable. Matrix metalloproteinases (MMPs) are enzymes involved in the development of periapical pathology, specifically inflammation and tissue destruction. To elucidate pathogenesis of periapical granulomas and radicular cysts, we undertook a detailed analysis of gene expression of MMP-1, MMP-2 and their tissue inhibitors, TIMP-1 and TIMP-2. METHODS: A total of 149 samples were analyzed using real-time PCR (59 radicular cysts, 50 periapical granulomas and 40 healthy gingiva samples as controls) for expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 genes. The determination of best reference gene for expression analysis of periapical lesions was done using a panel of 12 genes. RESULTS: We have shown that ß-actin and GAPDH are not the most stable reference controls for gene expression analysis of inflammatory periapical tissues and healthy gingiva. The most suitable reference gene was determined to be SDHA (a succinate dehydrogenase complex, subunit A, flavoprotein [Fp]). We found that granulomas (n = 50) and radicular cysts (n = 59) exhibited significantly higher expression of all four examined genes, MMP-1, MMP-2, TIMP-1, and TIMP-2, when compared to healthy gingiva (n = 40; P < 0.05). CONCLUSION: This study has confirmed that the expression of MMP-1, MMP-2, TIMP-1, and TIMP-2 genes is important for the pathogenesis of periapical inflammatory lesions. Since the abovementioned markers were not differentially expressed in periapical granulomas and radicular cysts, the challenge of finding the genetic differences between the two lesions still remains.

Bioinformatics, interaction network analysis, and neural networks to characterize gene expression of radicular cyst and periapical granuloma.

INTRODUCTION: Bioinformatics has emerged as an important tool to analyze the large amount of data generated by research in different diseases. In this study, gene expression for radicular cysts (RCs) and periapical granulomas (PGs) was characterized based on a leader gene approach. METHODS: A validated bioinformatics algorithm was applied to identify leader genes for RCs and PGs. Genes related to RCs and PGs were first identified in PubMed, GenBank, GeneAtlas, and GeneCards databases. The Web-available STRING software (The European Molecular Biology Laboratory [EMBL], Heidelberg, Baden-Württemberg, Germany) was used in order to build the interaction map among the identified genes by a significance score named weighted number of links. Based on the weighted number of links, genes were clustered using k-means. The genes in the highest cluster were considered leader genes. Multilayer perceptron neural network analysis was used as a complementary supplement for gene classification. RESULTS: For RCs, the suggested leader genes were TP53 and EP300, whereas PGs were associated with IL2RG, CCL2, CCL4, CCL5, CCR1, CCR3, and CCR5 genes. CONCLUSIONS: Our data revealed different gene expression for RCs and PGs, suggesting that not only the inflammatory nature but also other biological processes might differentiate RCs and PGs.

FOXP3 DNA methylation levels as a potential biomarker in the development of periapical lesions.

INTRODUCTION: Epigenetic mechanisms, such as DNA methylation, can modify gene expression patterns without changing the DNA sequence, comprising a tool that cells use to lock genes in the "off" position. Variations in the methylation profile have been correlated to a variety of human diseases. Here, we hypothesize that DNA methylation in immune response-related genes may contribute to the development of periapical lesions. METHODS: The DNA methylation patterns of 22 immune response-related gene promoters were evaluated in 137 human periapical granulomas, 8 apical cysts, and 31 healthy gingival tissues from 2 independent cohorts using a pathway-specific real-time polymerase chain reaction array (EpiTect Methyl II; Qiagen Inc, Valencia, CA). Messenger RNA expression analysis by qualitative polymerase chain reaction was also performed. SABiosciences's hierarchical clustering and methylation (Qiagen, Valencia, CA) and Prism6 software (GraphPad Software, Inc, La Jolla, CA) were used for data analysis. RESULTS: FOXP3 gene promoter showed the highest level of methylation in both periapical granulomas and apical cysts (P < .001), and methylation levels were inversely correlated with FOXP3 messenger RNA expression in the lesions. Furthermore, FOXP3 expression was prevalent in inactive lesions and was positively correlated with interleukin-10 and transforming growth factor beta levels. CONCLUSIONS: Our results suggest that FOXP3 acts as a master switch governing the development and function of T-regulatory cells, whose functions include the inhibition of immune responses and temper inflammation. The observed differential methylation patterns of FOXP3 in periapical lesions may be crucial in determining its suppressive activity and may be involved in periapical lesion development.

Methylation pattern of IFNG in periapical granulomas and radicular cysts.

INTRODUCTION: Interferon-γ plays an important role in the pathogenesis of periapical lesions, and the methylation of IFNG has been associated with transcriptional inactivation. The purpose of the present study was to investigate IFNG promoter methylation in association with gene transcription and protein levels in periapical granulomas and radicular cysts. METHODS: Methylation-specific polymerase chain reaction was used to assess the DNA methylation pattern of the IFNG gene in 16 periapical granulomas and 13 radicular cyst samples. The transcription levels of IFNG mRNA were verified by quantitative real-time polymerase chain reaction, and protein expression was evaluated by immunohistochemistry. RESULTS: All the periapical lesion samples exhibited partial or total methylation of the IFNG gene. In addition, an increased methylation profile was found in radicular cysts compared with periapical granulomas. Increased IFNG mRNA expression was observed in the partially methylated periapical lesion samples relative to the samples that were completely methylated. CONCLUSIONS: The present study provides the first evidence of the possible impact of IFNG methylation on IFNG transcription in periapical lesions.

Expression analysis of wound healing genes in human periapical granulomas of progressive and stable nature.

INTRODUCTION: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. METHODS: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). RESULTS: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001). CONCLUSIONS: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success.

Cell synthesis, proliferation and apoptosis in human dental periapical lesions analysed by in situ hybridisation and immunohistochemistry.

OBJECTIVE: The role of structural and host defensive cells in periapical lesions has been assessed previously by morphometric and immunohistochemical studies. The aim of this study was to investigate the function of peri- apical cells by employing molecular techniques to estimate the cell synthetic activity, proliferation and apoptosis in these lesions. We specifically sought answers to the following questions. Which cells of the periapical lesions are quiescent or actively synthesising proteins? Do immune cells proliferate in this region in the same way as epithelial cells proliferate? Furthermore do cells in peri- apical lesions undergo apoptosis, and if so which cells exhibit this programmed cell death? MATERIALS: Twenty-five periapical tissue samples (15 granulomas and 10 radicular cysts) were assessed. Poly-adenosine (poly (A)) RNA and ribosomal RNA (rRNA) bearing cells in formalin-fixed/paraffin-embedded peri- apical tissues were analysed by in situ hybridization (ISH) using digoxigenin-labelled oligo d (T) and 28S rRNA probes respectively in order to estimate cell synthetic activity. Furthermore, S-phase proliferating and cycling cells were examined by ISH using a histone probe and Ki-67 immunostaining so as to assess cellular proliferation. Mononuclear cells were further differentiated by immunohistochemistry (IHC) as T cells, B cells and macrophages. Apoptotic cells were determined by in situ end-labelling methodology for detecting fragmented DNA. RESULTS: Poly (A) RNA (mostly messenger RNA) and 28S rRNA-expressing cells were detected in all samples. Plasma cells exhibited strongest staining for the two probes, with slight to moderate staining found in the epithelium, fibroblasts, macrophages, endothelial cells and lymphocytes, whereas almost all polymorphonuclear leucocytes (PMN) were negative for these probes. A few histone mRNA-expressing cells were detected in basal and suprabasal epithelial cells and mononuclear cells in 15/25 cases but their reactivity was weak. Ki-67 positive cells were found in all samples and their numbers were generally higher than histone mRNA positive cells. Apo- ptotic cells were detected in 23/25 cases and the majority of apoptotic cells were PMN which were engulfed by large cytophagocytic macrophages. CONCLUSION: This study indicates that in dental periapical lesions, apoptosis occurs predominantly in PMN. It is evident that most cells apart from PMN are exhibiting synthetic activity but only epithelial cells undergo proliferation which implies that immune cells must proliferate at distant lymph nodes and travel to the periapical lesion rather than proliferating within the lesion. These results suggest considerable advantages in estimating gene expression within cells in addition to the immunohistochemical detection of cells to determine cell activity at inflamed sites. Clearly, functional cell synthetic activity, resolution and clearance systems operate in peri- apical cystic and granuloma lesions.

Quantification of PCNA+ cells within odontogenic jaw cyst epithelium.

The aim of this study was to investigate the reactivity of the epithelial linings of the three major types of odontogenic cyst with a monoclonal antibody to proliferating cell nuclear antigen (PCNA; clone PC10). PCNA expression was studied in odontogenic cysts (n = 31) and normal oral epithelium (n = 10) using a biotin-streptavidin method on routinely processed paraffin sections. PCNA+ cells were counted manually and related to the length of basement membrane (mm) and the epithelial area (mm2) as determined by TV image analysis. The epithelial linings of odontogenic keratocysts (OKC; n = 11) contained the highest number of PCNA+ cells, most of which were located in the suprabasal layers. The mean value of PCNA+ cells in OKC linings (94.4 +/- 22.7 cells/mm) was similar to that of oral epithelia (80.8 +/- 20.6 cells/mm), but both were significantly higher than that of dentigerous (n = 10, 5.1 +/- 3.0 cells/mm) and radicular (n = 10, 11.0 +/- 4.1 cells/mm) cyst linings (P < 0.005). The epithelial distribution of PCNA+ cells differed between groups with the basal/suprabasal PCNA+ cell ratio in OKC linings (0.05 +/- 0.02) being significantly lower than that of normal oral epithelium (0.5 +/- 0.14), dentigerous (1.6 +/- 1.23) and radicular (1.9 +/- 1.09) cyst linings respectively (P < 0.005). These results demonstrate differences in PCNA expression between the epithelial linings of the major odontogenic cyst types, indicating differences in proliferative and differentiation processes within these lesions.

Discrimination of parakeratinised odontogenic keratocysts from other odontogenic and non-odontogenic cyst types by expression of a 38kd cell-surface glycoprotein.

We have identified strong expression of a 38-kD cell surface glycoprotein (gp38), a marker of basal cell carcinomas (BCCs), in basal and suprabasal epithelial cell membranes of parakeratinised odontogenic keratocysts. In contrast, orthokeratinised cysts and most other odontogenic cyst types, ameloblastomas, normal stratified oral epithelium, cell rests of Malassez and glands of Serres, all proved negative. To our knowledge this is the first histochemical marker to distinguish between these major cyst types. It has obvious uses in the diagnosis of inflamed keratocysts and the separation of ameloblastomas from BCCs and may find a role in studies of the developmental biology of other odontogenic structures.