Comparative Metabolomics Reveals the Microenvironment of Common T-Helper Cells and Differential Immune Cells Linked to Unique Periapical Lesions.

Periapical abscesses, radicular cysts, and periapical granulomas are the most frequently identified pathological lesions in the alveolar bone. While little is known about the initiation and progression of these conditions, the metabolic environment and the related immunological behaviors were examined for the first time to model the development of each pathological condition. Metabolites were extracted from each lesion and profiled using gas chromatography-mass spectrometry in comparison with healthy pulp tissue. The metabolites were clustered and linked to their related immune cell fractions. Clusters I and J in the periapical abscess upregulated the expression of MMP-9, IL-8, CYP4F3, and VEGF, while clusters L and M were related to lipophagy and apoptosis in radicular cyst, and cluster P in periapical granuloma, which contains L-(+)-lactic acid and ethylene glycol, was related to granuloma formation. Oleic acid, 17-octadecynoic acid, 1-nonadecene, and L-(+)-lactic acid were significantly the highest unique metabolites in healthy pulp tissue, periapical abscess, radicular cyst, and periapical granuloma, respectively. The correlated enriched metabolic pathways were identified, and the related active genes were predicted. Glutamatergic synapse (16-20),-hydroxyeicosatetraenoic acids, lipophagy, and retinoid X receptor coupled with vitamin D receptor were the most significantly enriched pathways in healthy control, abscess, cyst, and granuloma, respectively. Compared with the healthy control, significant upregulation in the gene expression of CYP4F3, VEGF, IL-8, TLR2 (P < 0.0001), and MMP-9 (P < 0.001) was found in the abscesses. While IL-12A was significantly upregulated in cysts (P < 0.01), IL-17A represents the highest significantly upregulated gene in granulomas (P < 0.0001). From the predicted active genes, CIBERSORT suggested the presence of natural killer cells, dendritic cells, pro-inflammatory M1 macrophages, and anti-inflammatory M2 macrophages in different proportions. In addition, the single nucleotide polymorphisms related to IL-10, IL-12A, and IL-17D genes were shown to be associated with periapical lesions and other oral lesions. Collectively, the unique metabolism and related immune response shape up an environment that initiates and maintains the existence and progression of these oral lesions, suggesting an important role in diagnosis and effective targeted therapy.

Salivary Metabolomics Fingerprint of Chronic Apical Abscess with Sinus Tract: A Pilot Study.

Chronic apical abscess (CAA) is a lesion of apical periodontitis mostly characterized by areas of liquefactive necrosis with disintegrating polymorphonuclear neutrophils surrounded by macrophages. Its presence leads to local bacterial infection, systemic inflammatory response, pain, and swelling. The use of a novel approach for the study of CAA, such as metabolomics, seems to be important since it has proved to be a powerful tool for biomarkers discovery which could give novel molecular insight on CAA. So, the aim of this study was to verify the possibility to identify the metabolic fingerprint of CAA through the analysis of saliva samples. Nineteen patients were selected for this study: eleven patients affected by CAA with a sinus tract constituted the study group whereas eight patients without clinical and radiographic signs of CAA formed the healthy control group. Saliva samples were collected from each subject and immediately frozen at -80°C. Metabolomic profiles were obtained using a gas chromatography/mass spectrometry instrument. Subsequently, in order to compare the two groups, a multivariate statistical model was built that resulted to be statistically significant. The class of metabolites characterizing the CAA patients was closely related to the bacterial catabolism, tissue necrosis, and presence of a sinus tract. These preliminary results, for the first time, indicate that saliva samples analyzed by means of GC/MS metabolomics may be useful for identifying the presence of CAA, leading to new insights into this disease.

Mesenchymal Stem Cell Marker Expression in Periapical Abscess.

INTRODUCTION: There is evidence that acute periapical lesions present a greater potential for cyst formation. Recently, it was found that these lesions have cells with characteristics of pluripotent stem cells, which may influence cyst development. However, a more complete phenotype investigation of stem cells in a specific sample of periapical abscesses is required. The aim of this study was to analyze the immunohistochemical expression of mesenchymal stem cell (MSC) markers in periapical abscesses and to evaluate differences in their expression in relation to acute and chronic periapical lesions. METHODS: Immunohistochemistry was used to access MSC marker expression (CD44, CD73, and CD105) in samples of periapical abscesses (n = 10), granulomas (n = 10), cysts (n = 10), and apical papillae (n = 10). Immunohistochemical expression was evaluated by a quantitative scoring system. The chi-square test was used to assess the association between MSC marker expression and the histopathological diagnosis at a 5% significance level. RESULTS: CD44 and CD73 immunostaining was observed in mesenchymal cells located in the outer portion of the abscess and periapical cyst specimens. CD105 immunoexpression was found predominantly in mesenchymal and vascular endothelial cells of the lesions studied. MSC marker expression was higher in the periapical abscesses, with a significant association between MSCs and the histopathological diagnosis of an abscess (P < .05). CONCLUSIONS: The periapical region is a rich source of MSCs. The greater presence of MSCs in periapical abscesses found in this study could hold an important clue into understanding the pathological pathway of periapical cyst formation.

Human Exoproteome in Acute Apical Abscesses.

INTRODUCTION: An acute apical abscess is a severe response of the host to massive invasion of the periapical tissues by bacteria from infected root canals. Although many studies have investigated the microbiota involved in the process, information on the host factors released during abscess formation is scarce. The purpose of this study was to describe the human exoproteome in samples from acute apical abscesses. METHODS: Fourteen pus samples were obtained by aspiration from patients with an acute apical abscess. Samples were subjected to protein digestion, and the tryptic peptides were analyzed using a mass spectrometer and ion trap instrument. The human proteins identified in this analysis were classified into different functional categories. RESULTS: A total of 303 proteins were identified. Most of these proteins were involved in cellular and metabolic processes. Immune system proteins were also very frequent and included immunoglobulins, S100 proteins, complement proteins, and heat shock proteins. Polymorphonuclear neutrophil proteins were also commonly detected, including myeloperoxidases, defensins, elastases, and gelatinases. Iron-sequestering proteins including transferrin and lactoferrin/lactotransferrin were found in many samples. CONCLUSIONS: The human exoproteome included a wide variety of proteins related to cellular processes, metabolism, and immune response. Proteins involved in different mechanisms against infection, tissue damage, and protection against tissue damage were identified. Knowledge of the presence and function of these proteins using proteomics provides an insight into the complex host-pathogen relationship, the host antimicrobial strategies to fight infections, and the disease pathogenesis.

Analysis of Interleukin 17A in periapical abscess and granuloma lesions.

Interleukin 17A (IL-17A) is a proinflammatory cytokine responsible for the initiation and propagation of inflammation. One of its actions is the recruitment of neutrophils to the site of infection. The aim of this study was to investigate whether there is association between IL-17A expression and neutrophil infiltration in periapical abscesses and periapical granulomas, as well as to find which type of T lymphocyte effector (CD4+ or CD8+) expresses IL-17A in these lesions. Elastase, CD4, CD8, and IL-17A were analyzed by immunohistochemistry and immunofluorescence, in the biopsies of periapical lesions. Abscess lesions exhibited the highest labeling area for IL-17A (p = 0.011). During double immunofluorescence staining, there were significantly more CD4+/IL-17A+ cells compared to CD8+/IL-17A+ cells, both in the abscesses (p = 0.025) and granulomas (p = 0.011). In conclusion, IL-17A was intensively expressed in periapical abscesses rich in neutrophils. The high percentage of IL-17A in these cases suggests the participation of this cytokine particularly in the acute stages of the inflammatory process of the periapical lesions.

Analysis of Interleukin 17A in periapical abscess and granuloma lesions

Abstract Interleukin 17A (IL-17A) is a proinflammatory cytokine responsible for the initiation and propagation of inflammation. One of its actions is the recruitment of neutrophils to the site of infection. The aim of this study was to investigate whether there is association between IL-17A expression and neutrophil infiltration in periapical abscesses and periapical granulomas, as well as to find which type of T lymphocyte effector (CD4+ or CD8+) expresses IL-17A in these lesions. Elastase, CD4, CD8, and IL-17A were analyzed by immunohistochemistry and immunofluorescence, in the biopsies of periapical lesions. Abscess lesions exhibited the highest labeling area for IL-17A (p = 0.011). During double immunofluorescence staining, there were significantly more CD4+/IL-17A+ cells compared to CD8+/IL-17A+ cells, both in the abscesses (p = 0.025) and granulomas (p = 0.011). In conclusion, IL-17A was intensively expressed in periapical abscesses rich in neutrophils. The high percentage of IL-17A in these cases suggests the participation of this cytokine particularly in the acute stages of the inflammatory process of the periapical lesions.

Effects of induced periapical abscesses on rat pregnancy outcomes.

OBJECTIVE: The objective of this study was to determine the effects of induced periapical abscesses on pregnant rats. DESIGN: In 1/2 of the animals (n=16), the pulps of the maxillary right molars were exposed to the oral environment, which resulted in a periapical abscess. The other 1/2 (n=16) were sham-operated. 1/2 of the animals of both groups became pregnant 2 weeks later. The pregnancy duration, and weight and number of pups were assessed at delivery. Serum, liver and uterine horn samples were taken from all animals at euthanasia and serum IL-6, endothelin-1, TNF-alpha, IL-10, cortisol and insulin were determined by ELISA. Liver concentrations of IL-6, CRP and IL-6 and uterine horn concentrations of IL-6, vascular endothelial growth factor (VEGF), TNF-alpha, IL-10 and IL-1-beta were assessed by ELISA. Blood glucose concentrations were determined using a glucometer. Outcome variables were compared by factorial ANOVA, a post hoc Tukey test, and Pearson's correlation test. RESULTS: Pregnant rats with periapical abscesses had a significantly longer pregnancy and delivered pups with a significantly higher birthweight (p<0.05). They had significantly higher concentrations of IL-6, VEGF, IL-1-beta, and IL-10 within the uterine horn and IL-6, CRP and TNF-alpha within the liver (p<0.01). Blood glucose and serum TNF-alpha, IL-6, endothelin-1, IL-10, and insulin concentrations were significantly higher in the pregnant animals with pulpal abscesses (p<0.01). CONCLUSION: The significant increase in serum TNF-alpha, taken together with significant increases in blood glucose and serum insulin concentrations, suggest that animals with induced periapical abscesses developed insulin resistance, which significantly affected their pregnancy outcomes.

Induction of cyclooxygenase-2 mRNA and protein expression by epoxy resin and zinc oxide-eugenol based root canal sealers in human osteoblastic cells.

An ideal root canal sealer should be nonirritating to the surrounding tissues. Unfortunately, all histological investigation demonstrated that all types of root canal sealer can induce mild to severe inflammatory alternations. However, there is little information on the precise mechanisms about root canal sealers-induced inflammatory reaction. Cyclooxygenase-2 (COX-2) is an inducible enzyme believed to be responsible for prostaglandin synthesis at site of inflammation. The aim of the present study was to investigate the effects of epoxy resin (AH26) and zinc oxide-eugenol based (Endomethansone and N2) root canal sealers on the expression of COX-2 mRNA gene and protein in cultured human osteoblastic cells. Investigations of the time dependence of COX-2 mRNA expression in root canal sealer-treated human osteoblastic cells revealed a rapid accumulation of the transcript, a significant signal first detectable within 2h and diminished to control level after 24h. In addition, all root canal sealers also induced COX-2 protein expression in human osteoblastic cells. Furthermore, to elucidate whether induction of COX-2 is associated with cytotoxicity, NS-398 (a selective COX-2 inhibitor), was added to test its protective effects. NS-398 at non-cytotoxic dose is not able to prevent root canal sealers-induced cytotoxicity. Taken together, the activation of COX-2 expression may be one of the pathogenesis of root canal sealers-induced periapical inflammation. In addition, root canal sealers-induced cytotoxicity is not directly via the induction of COX-2 expression.

Balance of interleukin-1 beta and interleukin-1 receptor antagonist in human periapical lesions.

Interleukin-1 beta (IL-1 beta) has been considered as a major potent mediator of bone resorption and implicated in the development of human periapical lesions. Among naturally occurring interleukin-1 (IL-1) inhibitors, IL-1 receptor antagonist (IL-1ra) is a 22 kDa protein that shares homology with IL-1 beta and IL-1 alpha, binds to IL-1 receptor with similar affinity to IL-1, and has no known agonist properties. In this study, we measured the periapical exudate (PE) levels of IL-1 beta and IL-1ra from human periapical lesions. PE samples were collected from root canals during routine endodontic treatments, and the enzyme-linked immunosorbent assay was used to measure PE-IL-1 beta and IL-1ra. Detectable levels of both IL-1 beta and IL-1ra were found in 25 of 29 clinical samples. Relatively high levels of IL-1ra compared with IL-1 beta (mean IL-1ra:IL-1 beta ratio = 128:7; range: 0.9 to 495.4), and significantly positive correlation between IL-1ra and IL-1 beta levels was found. The PE-IL-1ra:IL-1 beta ratios obtained from symptomatic lesions were significantly lower than those from asymptomatic lesions. These results suggest that IL-1ra-mediated IL-1 antagonism occurred to block locally produced IL-1 activity, and the balance of IL-1 to IL-1ra production may be crucial in the development of periapical lesions.

Immunohistochemical detection of prostaglandins E2, F2 alpha, and 6-keto-prostaglandin F1 alpha in experimentally induced periapical inflammatory lesions in rats.

The immunohistochemical localization of prostaglandin (PG) E2, PGF2 alpha, and 6-keto-PGF1 alpha (a stable metabolite of PGI2) was demonstrated in rat periapical inflammatory lesions induced by opening the pulp chamber. Two wk postoperatively, suppurative periapical lesions were formed, and active bone resorption was seen surrounding these lesions. Immunohistochemical examination showed that macrophages infiltrating in inflammatory tissue were positively stained for the examined PGs. In some lesions, wherein acute inflammatory changes subsided and proliferation of fibroblasts started, the fibroblasts were positively stained for 6-keto-PGF1 alpha. Osteocytes and osteoblasts were also positive for 6-keto-PGF1 alpha not only in experimental animals, but also in untreated animals. However the staining intensity of the PG in these cells was higher in periapical lesions than in normal condition. These findings suggested that the cellular sources of the PGs in the periapical lesions are mainly macrophages and fibroblasts, and that the PGs produced by these cells, and possibly osteoblast and osteocytes, may contribute to the osteolytic resorption of periapical lesions.

The importance of immunoglobulin-breakdown supporting the growth of bacteria in oral abscesses.

Oral bacteria play an important rôle in the causation of oro-facial abscesses. However, they can also be involved in brain, liver and lung abscesses. To persist, it is essential that these bacteria can grow on those sites. The main source of nutrients for growth in abscesses is likely to be tissue exudate, which is rich in serum-derived proteins, and relatively poor in free amino acids and carbohydrates. Degradation of intact proteins seems a crucial step in providing the peptides necessary for energy generation. The aim of this study was to investigate the capacity of microorganisms from asscesses to degrade serum proteins, in particular immunoglobulins. To this end, samples were taken by aspiration from 16 odontogenic abscesses. It was found that pus from abscesses differed strongly in the concentration of viable bacterial cells. The ability of the abscess microflora to degrade serum proteins was investigated after growth of the sample in heat-inactivated human serum. The microflora from abscesses with a high concentration (n = 10) of bacteria strongly degraded immunoglobulins, whereas breakdown of immunoglobulins was virtually absent after growth of the microflora from low-bacterial concentration (n = 6) abscesses. Bacteriological analyses revealed the presence of at least one proteinase-producing species, like Porphyromonas, black-pigmented Prevotella species, or Actinomyces meyeri, in abscesses with a high density of bacteria, but not in those with low bacterial density. The results indicate that the capacity to degrade intact proteins, in particular immunoglobulins, is a major determinant of bacterial growth in abscesses.

Periapical destructions caused by experimental pulpal inoculation of Streptococcus mutans in rats.

The development of pulpal and periapical changes in rat molars was studied after inoculation of Streptococcus mutans (GS-5) into the pulp chamber. Before injection into the pulp Streptococcus mutans was cultured on a special collagen substrate and "trained" to break down collagen. The destruction of the alveolar bone periapically could be demonstrated both roentgenologically and histopathologically. Large numbers of inflammatory cells in the pulp chamber and the periapical area, as well as carious dentin, were present. The pulpally inoculated bacteria could also be recovered from the systemic blood. The identity between the pulpally inoculated bacteria and the bacteria recovered from the blood was proved by gel precipitation.

Occurrence of amyloid in the teeth-supporting tissues of patients with rheumatoid diseases. An immunohistochemical study.

An investigation was carried out to determine whether amyloid could be detected in the dental periapical lesions or gingival buccal fold, or in both, of patients with rheumatoid disease but in whom amyloidosis had not been diagnosed. Tissue from the dental periapical lesions of 36 rheumatoid and 23 control patients, as well as from the gingival buccal fold of 11 of the rheumatoid and 11 of the control patients, were examined by the direct immunofluorescence technique and by polarization microscopy of sections stained with Congo red. Amyloid was observed almost 5 times more often, or in 19% of the dental periapical lesions of rheumatoid patients: it was detected by immunofluorescence in 7 rheumatoid patients and 1 control patient, and by polarization microscopy in 6 of these 7 rheumatoid patients as well as in the 1 control. Rectal biopsies performed on these 8 patients were positive in only 1 of the rheumatoid patients. Amyloid was not detected in any of the gingival biopsies. Serum samples from the rheumatoid and the control patients were tested for the presence of the nonimmunoglobulin amyloid fibril-related serum component, protein ASC. Nineteen of the 36 rheumatoid patients, including the 7 whose dental periapical lesions contained amyloid, had protein ASC in their serum.

Free rheumatoid factor in dental periapical lesions and gingivae of patients with rheumatoid disease.

To determine whether evidence of rheumatoid inflammation, in the form of free rheumatoid factor, might be found in the teeth-supporting tissues of patients with known rheumatoid disease, tissues from the dental periapical lesions of one group of 50 rheumatoid and 23 control patients, and from the marginal gingivae of a second group of 58 rheumatoid patients were examined by the direct immunofluorescence technique that employed fluroesceinisothiocyanate (FITC)-labelled aggregated human IgG. The gingival tissues contained no free rheumatoid factor. Free rheumatoid factor-producing plasma cells were, however, detected in the dental periapical lesions of 3 of the 50 rheumatoid patients, i.e. in 6%, and in 1 of the control patients i.e., in 4%. This control patient had suffered from nephritis 10 months prior to the investigation. Because free rheumatoid factor did occur, albeit infrequently, in the dental periapical lesions of rheumatoid patients, a search for IgG rheumatoid factor, known to occur in greater abundance than the IgM type although "hidden", was indicated.