Human Gingival Samples Research Articles

Modulating the sEH/EETs Axis Restrains Specialized Proresolving Mediator Impairment and Regulates T Cell Imbalance in Experimental Periodontitis.

Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids are short-acting lipids involved in resolution of inflammation. Their short half-life, due to its metabolism by soluble epoxide hydrolase (sEH), limits their effects. Specialized proresolving mediators (SPMs) are endogenous regulatory lipids insufficiently synthesized in uncontrolled and chronic inflammation. Using an experimental periodontitis model, we pharmacologically inhibited sEH, examining its impact on T cell activation and systemic SPM production. In humans, we analyzed sEH in the gingival tissue of periodontitis patients. Mice were treated with sEH inhibitor (sEHi) and/or EETs before ligature placement and treated for 14 d. Bone parameters were assessed by microcomputed tomography and methylene blue staining. Blood plasma metabololipidomics were carried out to quantify SPM levels. We also determined T cell activation by reverse transcription-quantitative PCR and flow cytometry in cervical lymph nodes. Human gingival samples were collected to analyze sEH using ELISA and electrophoresis. Data reveal that pharmacological sEHi abrogated bone resorption and preserved bone architecture. Metabololipidomics revealed that sEHi enhances lipoxin A4, lipoxin B4, resolvin E2, and resolvin D6. An increased percentage of regulatory T cells over Th17 was noted in sEHi-treated mice. Lastly, inflamed human gingival tissues presented higher levels and expression of sEH than did healthy gingivae, being positively correlated with periodontitis severity. Our findings indicate that sEHi preserves bone architecture and stimulates SPM production, associated with regulatory actions on T cells favoring resolution of inflammation. Because sEH is enhanced in human gingivae from patients with periodontitis and connected with disease severity, inhibition may prove to be an attractive target for managing osteolytic inflammatory diseases.

Transcriptome analysis of differentially expressed genes involved in the inflammageing status of gingiva in aged mice.

To identify immune-inflammation-related genes related to susceptibility to periodontitis in the gingiva of aged mice with RNA sequencing. Gingival samples from 18-month-old, 8-week-old healthy mice and 8-week-old mice with periodontitis were taken for RNA-seq. The differentially expressed genes (DEGs) were validated with qRT-PCR using mouse and human gingival samples. 977 (upregulated) and 1824 (downregulated) genes were identified in the old compared with the young mice. 14.2% were related to immune-inflammatory responses. This proportion of overlap (ageing and periodontitis)-DEGs was higher (48.4%). Enrichment analysis of overlap (ageing and periodontitis)-DEGs showed that the top five GO and KEGG terms were related to the immune-inflammatory responses, and disease analysis was more specific to periodontitis. The candidate genes of overlap (ageing and periodontitis)-DEGs selected by protein-protein interaction (PPI) network showed the higher match with clinical data sets. By qRT-PCR, nine candidate genes were identified as hub genes that are associated with susceptibility to periodontitis in the elderly, including CXCL3, CXCL5, CSF3, CSF3R, FPR1, IL1B, OSM, SERPINE1 and SELP. Our studies provide insights into the mechanisms by which ageing affects the immune-inflammatory status of gingival tissues, thereby increasing the risk of periodontitis. It may become targets for future prevention of periodontitis.

The angiotensin converting enzyme 2/angiotensin-(1-7)/Mas Receptor axis as a key player in alveolar bone remodeling.

The renin-angiotensin system (RAS), aside its classical hormonal properties, has been implicated in the pathogenesis of inflammatory disorders. The angiotensin converting enzyme 2/angiotensin-(1-7)/Mas Receptor (ACE2/Ang-(1-7)/MasR) axis owns anti-inflammatory properties and was recently associated with bone remodeling in osteoporosis. Thus, the aim of this study was to characterize the presence and effects of the ACE2/Ang-(1-7)/MasR axis in osteoblasts and osteoclasts in vitro and in vivo. ACE2 and MasR were detected by qPCR and western blotting in primary osteoblast and osteoclast cell cultures. Cells were incubated with different concentrations of Ang-(1-7), diminazene aceturate (DIZE - an ACE2 activator), A-779 (MasR antagonist) and/or LPS in order to evaluate osteoblast alkaline phosphatase and mineralized matrix, osteoclast differentiation and cytokine expression, and mRNA levels of osteoblasts and osteoclasts markers. An experimental model of alveolar bone resorption triggered by dysbiosis in rats was used to evaluate bone remodeling in vivo. Rats were treated with Ang-(1-7), DIZE and/or A-779 and periodontal samples were collected for immunohistochemistry, morphometric analysis, osteoblast and osteoclast count and cytokine evaluation. Human gingival samples from healthy and periodontitis patients were also evaluated for detection of ACE2 and MasR expression. Osteoblasts and osteoclasts expressed ACE2 and MasR in vitro and in vivo. LPS stimulation or alveolar bone loss induction reduced ACE2 expression. Treatment of bone cells with Ang-(1-7) or DIZE stimulated osteoblast ALP, matrix synthesis, upregulated osterix, osteocalcin and collagen type 1 transcription, reduced IL-6 expression, and decreased osteoclast differentiation, RANK and IL-1β mRNA transcripts, and IL-6 and IL-1β levels, in a MasR-dependent manner. In vivo, Ang-(1-7) and DIZE decreased alveolar bone loss through improvement of osteoblast/osteoclast ratio. A-779 reversed such phenotype. ACE2/Ang-(1-7)/MasR axis activation reduced IL-6 expression, but not IL-1β. ACE2 and MasR were also detected in human gingival samples, with higher expression in the healthy than in the inflamed tissues. These findings show that the ACE2/Ang-(1-7)/MasR is an active player in alveolar bone remodeling.

Interleukin-33 and RANK-L Interplay in the Alveolar Bone Loss Associated to Periodontitis.

IntroductionChronic Periodontitis (CP) is an inflammatory disease of bacterial origin that results in alveolar bone destruction. Porphyromonas gingivalis (Pg), one of the main periopathogens, initiates an inflammatory cascade by host immune cells thereby increasing recruitment and activity of osteoclasts, the bone resorbing cells, through enhanced production of the crucial osteoclastogenic factor, RANK-L. Antibodies directed against some cytokines (IL-1β, IL-6 and TNF-α) failed to exhibit convincing therapeutic effect in CP. It has been suggested that IL-33, could be of interest in CP.Objectivethe present study aims to analyze whether and how IL-33 and RANK-L and/or their interplay are involved in the bone destruction associated to CP.Material and MethodsmRNAs and protein expressions of IL-33 and RANK-L were analyzed in healthy and CP human gingival samples by immunohistochemistry (IHC) and RT-qPCR. Murine experimental periodontitis (EP) was induced using Pg infected ligature and Pg free ligature around the first maxillary molar. Alveolar bone loss was recorded by μCT. Mouse gingival explants were stimulated for 24 hours with IL-33 and RANK-L mRNA expression investigated by RT-qPCR. Human oral epithelial cells were infected by Pg for 6, 12; 24 hours and IL-33 and RANK-L mRNA expressions were analyzed by RT-qPCR.ResultsIL-33 is overexpressed in gingival epithelial cells in human affected by CP as in the murine EP. In human as in murine gingival cells, RANK-L was independently induced by Pg and IL-33. We also showed that the Pg-dependent RANK-L expression in gingival epithelial cells occured earlier than that of IL-33.ConclusionOur results evidence that IL-33 overexpression in gingival epithelial cells is associated with CP and may trigger RANK-L expression in addition to a direct effect of Pg. Finally, IL-33 may act as an extracellular alarmin (danger signal) showing proinflammatory properties in CP perpetuating bone resorption induced by Pg infection.

Class I and II histone deacetylase expression in human chronic periodontitis gingival tissue.

Histone deacetylase inhibitors (HDACi) are being considered to treat chronic inflammatory diseases at low doses. Currently HDACi that are more specific are being developed to target particular HDACs; therefore, this study aimed to determine levels and distribution of class I and II HDAC in human gingival samples obtained from patients with chronic periodontitis. Gingival biopsies were obtained from patients with and without (mild inflammation, no bone loss) periodontitis. Total RNA was isolated for real-time quantitative polymerase chain reaction to determine expression of HDACs 1-10. Immunohistochemistry was used to determine protein distribution of HDACs 1, 5, 8 and 9. Factor VIII, CD3 and tartrate resistant acid phosphatase (TRAP) were detected in serial sections to identify blood vessels, lymphocytes, pre-osteoclasts and osteoclasts cells respectively. Tumour necrosis factor α (TNF-α) expression was also assessed. mRNA for HDAC 1, 5, 8 and 9 were significantly upregulated inchronic periodontitis gingival tissues compared to non-periodontitis samples(p<0.05). Significantly higher HDAC 1 protein expression was observed in chronic periodontitis samples (p<0.05), and was associated withCD3, TRAP and TNF-α-positive cells. HDAC 1, 5, 8 and 9 were expressed strongly by the factor VIII-positive microvasculature in the chronic periodontitis gingival tissues. HDAC 1, 5, 8 and 9 expression was higher in gingival tissues from patients with chronic periodontitis compared to non-periodontitis samples. Results suggest that these HDACs could therefore be targeted with specific acting HDACi.

Immunoexpression of vascular endothelial growth factor and Ki-67 in human gingival samples: An observational study.

Aim:To evaluate immunohistochemically vascular endothelial growth factor (VEGF) and Ki-67 in human gingival samples and to compare these factors between healthy and diabetic patients.Materials and Methods:A total of 50 subjects were included in the study. They were categorized into three groups: Periodontally healthy group, periodontally diseased gingiva without any systemic disease group and periodontally diseased gingiva with controlled type II diabetes mellitus (DM) group. Gingival biopsies were performed and immunohistochemical analysis were done for VEGF and Ki-67 staining in gingival samples.Results:The present study found moderate intensity staining for VEGF in periodontitis group and periodontitis with controlled type II DM group and mild intensity staining for VEGF in periodontally healthy group. With regard to Ki-67, negative staining was observed in periodontally healthy group and mild staining in periodontitis group and periodontitis with controlled type II DM group.Conclusion:Further investigation needs to be conducted to identify how VEGF and Ki-67 are involved in the tissue inflammation associated processes and the relationship between VEGF and Ki-67 in progression of periodontitis.

Induction of β-Defensin Expression by &lt;i&gt;Porphyromonas gingivalis&lt;/i&gt;-Infected Human Gingival Graft Transplanted in &lt;i&gt;nu/nu&lt;/i&gt; Mouse Subdermis

It is important to understand the onset of periodontal disease in terms of bacterial infection and host factors. Host-bacteria interactions can be elicited in human cultured cells and animal models, but these models provide only limited biological information about human host reactions against bacterial attacks. Development of an in vivo model using human gingival tissue is needed. We established an in vivo model using nu/nu mice and evaluated host defense following bacterial infection in human gingiva. Human gingival samples were collected from periodontitis patients and transplanted in nu/nu mouse subdermis. After 2 weeks, human characteristics were confirmed by positive immunohistochemical reactions for human-specific markers. We used this model to investigate human β-defensin-2 (hBD-2), an antimicrobial peptide that contributes to initial defense against bacterial invasion. Using real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry, we investigated whether hBD-2 expression was induced in human gingiva as a response to Porphyromonas gingivalis as a periodontal pathogen. Two hours after infection with bacteria, we detected increased expression of hBD-2 mRNA, which was localized in the epithelium of human gingiva. Using our in vivo model, we concluded that increased hBD-2 may play an important role in early defense from bacterial infection in human gingival epithelium.

Expression of angiogenesis-stimulating factors (VEGF, CD31, CD105) and angiogenetic index in gingivae of patients with chronic periodontitis

The aim of this study was to determine the prognostic value of the angiogenesis rate in chronic periodontitis (CP). A total of 61 human gingival samples were taken from patients with CP (n = 40) obtained during open curettage with gingivectomy, healthy periodontia (n = 15), and reactive lymph nodes (n = 7). Quantitative immunocytochemistry studies of VEGF, CD31 (PECAM-1) and CD105 (endoglin) were performed using the spatial visualization method. CD105/CD31 and VEGF/CD31 angiogenetic ratios (ARs) were established to determine the proliferation fraction of the endothelium. In patients with CP, the proliferation of blood vessels was observed, including the presence of numerous high endothelial venules (HEVs) and ordinary vessels. In gingival HEVs of patients with CP, the higher expression was shown by CD31 and, in turn CD105 and VEGF. The entire vascular expression of CD31 in the gingiva correlates with grading in lamina propria, but our study failed to document correlations between the expression of VEGF and CD105 and clinical data of patients with CP. Higher ARs were seen in gingivae of CP patients compared to controls. We concluded that overexpression of the angiogenesis-associated factors in CP suggests its significance in protracting the inflammatory process or periodic exacerbations of the process and destruction of the periodontium. The increased CD105/CD31 and VEGF/CD31 ratios in gingiva confirms an augmented proliferative fraction of the endothelium in gingiva with CP.

Expression of angiogenesis-stimulating factors (VEGF, CD31, CD105) and angiogenetic index in gingivae of patients with chronic periodontitis

The aim of this study was to determine the prognostic value of the angiogenesis rate in chronic periodontitis (CP). A total of 61 human gingival samples were taken from patients with CP (n = 40) obtained during open curettage with gingivectomy, healthy periodontia (n = 15), and reactive lymph nodes (n = 7). Quantitative immunocytochemistry studies of VEGF, CD31 (PECAM-1) and CD105 (endoglin) were performed using the spatial visualization method. CD105/CD31 and VEGF/CD31 angiogenetic ratios (ARs) were established to determine the proliferation fraction of the endothelium. In patients with CP, the proliferation of blood vessels was observed, including the presence of numerous high endothelial venules (HEVs) and ordinary vessels. In gingival HEVs of patients with CP, the higher expression was shown by CD31 and, in turn CD105 and VEGF. The entire vascular expression of CD31 in the gingiva correlates with grading in lamina propria, but our study failed to document correlations between the expression of VEGF and CD105 and clinical data of patients with CP. Higher ARs were seen in gingivae of CP patients compared to controls. We concluded that overexpression of the angiogenesis-associated factors in CP suggests its significance in protracting the inflammatory process or periodic exacerbations of the process and destruction of the periodontium. The increased CD105/CD31 and VEGF/CD31 ratios in gingiva confirms an augmented proliferative fraction of the endothelium in gingiva with CP.

Spontaneous tumorigenicity of primary human oral keratinocytes with human papillomavirus negativity and impaired apoptosis

Although >60% of oral cancer cases are not related to human papillomavirus (HPV) infection, most studies of oral carcinogenesis in human cells in vitro are carried out with human oral keratinocytes immortalized by HPV DNA. To explore whether human oral keratinocytes can spontaneously transform without HPV infection, we attempted to establish spontaneously immortalized and tumorigenic-transformed human oral keratinocytes by serial subculture to the post-mitotic stage. Here we report two spontaneously transformed human oral keratinocyte lines from adult human gingival samples. These lines were obviously immortal (>140 passages) and transformed phenotypes in vitro. One of the lines, Spi-HOK1, remained non-tumorigenic in nude mice, whereas the other line, Spt-HOK80, showed tumorigenicity. These lines showed epithelial origi-nality, but did not contain high-risk types of HPV DNAs. On karyotyping, Spi-HOK1 was aneuploid with a unique stable marker chromosome. Both cell lines revealed a mutation in the p53 gene, loss of p21WAF1/Cip1 and overexpression of p-Rb-Ser807/811. These cell lines were resistant to cisplatin-induced apoptosis by suppressing induction of apoptotic proteins. These results clearly demonstrate that spontaneous immortalization and spontaneous tumorigenic transformation of primary human oral keratinocytes can occur in vitro without HPV infection and are associated with chromosomal alterations, p53 mutation and impaired apoptosis. To our knowledge, this is the first report demonstrating that the Spi-HOK1 and Spt-HOK80 lines are novel cell lines that are spontaneously transformed from primary human oral keratinocytes.

Localization and quantification of TXB2 in human healthy and inflammatory gingival mucosa.

We conducted a study to localize and quantify the thromboxane B2 (TXB2) in human gingival tissue obtained from clinically healthy sites and patients with gingivitis and periodontitis. Human gingival samples were assayed for TXB2 by immunofluorescence on slides and enzyme-immunoassay (EIA). We found that concentrations of TXB2 in gingivitis sites are at mean 10-fold higher than in the healthy sites and histologically show a consistently intracytoplasmic staining with a significant increase in gingivitis. This argues in favour of a local production of TXB2. Concentrations of TXB2 at periodontitis sites are only 3-fold higher than in healthy sites and histologically show a stronger staining as for the gingivitis sections, with principally an extracellular localization. Thus, TXB2 could be released in large quantities in the crevicular fluid when the periodontitis stage has reached. These data suggest that the epithelial cells of the gingival tissue are involved in synthesis and secretion of TXB2, which occurred during the development of gingival inflammation.

Effect of epinephrine on prostaglandin E 2 and cyclic AMP levels in gingiva of patients with chronic periodontitis

The levels of prostaglandin. E2 (PGE 2) and cyclic AMP (cAMP) in human gingival samples were determined by means of radioimmunoassay after incubation with epinephrine (10- 6M) at different time intervals. The levels of PGE 2 and cAMP reached a maximum in the control and hormone treated samples after 80 and 40 minutes, respectively. Epinephrine seemed to affect a lesser increase in PGE 2 and a greater increase in cAMP values in gingival tissues when compared with control samples. When the data were analysed, there was statistically significant differences found between the control and hormone treated samples regarding both PGE 2 and cAMP.