Periapical lesions in two inbred strains of rats differing in immunological reactivity.

AIM: To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. METHODOLOGY: Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21 days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were <.05. RESULTS: DA rats developed significantly larger (p < .05) periapical lesions compared to AO rats as confirmed by radiographic and pathohistological analysis. The immunohistochemical staining intensity for CD3 was significantly greater in periapical lesions of DA rats compared to AO rats (p < .05). In DA rats, periapical lesions had a significantly higher (p < .05) percentage of CD3+ cells compared to AO rats. Also, the percentage of INF-γ, IL-17 and IL-10 CD3+CD4+ cells was significantly higher in DA rats (p < .05). DA rats had a significantly higher Th17/Th10 ratio. RT-PCR expression of IL-1ß, INF-γ and IL-17 genes was significantly higher in periapical lesions of DA compared to AO rats (p < .05). The receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin ratio was higher in DA compared to AO rats with periapical lesions (p < .05). Systemic levels of TNF-α and IL-6 were significantly higher in DA compared to AO rats (p < .05). Levels of lipid peroxidation measured as thiobarbituric acid reactive substances and reduced glutathione were significantly higher (p < .05) in the supernatant in the periapical lesions of DA rats. CONCLUSION: After pulp exposure, DA rats developed much larger periapical lesions compared to AO rats. Genetically determined differences in immunopathology have been demonstrated to be a significant element defining the severity of periapical lesions.

Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

BACKGROUND: Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. METHODS: Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. RESULTS: The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P < 0.05), while the numbers of IL-33- and ST2-positive endothelial cells were similar (both P > 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. CONCLUSIONS: IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis.

Human stem cell research and regenerative medicine--present and future.

INTRODUCTION: Stem cells are cells with the ability to grow and differentiate into more than 200 cell types. SOURCES OF DATA: We review here the characteristics and potential of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and adult stem cells (ASCs). AREAS OF AGREEMENT: The differentiation ability of all stem cell types could be stimulated to obtain specialized cells that represent renewable sources of functional cells useful for cell-based therapy. AREAS OF CONTROVERSY: The proof of functional differentiated cells needs to be investigated in more detail using both in vitro and in vivo assays including animal disease models and clinical studies. GROWING POINTS: Much progress has been made in the ASCs-based therapies. Meanwhile hESCs and iPSCs have dramatically emerged as novel approaches to understand pathogenesis of different diseases. AREAS TIMELY FOR DEVELOPING RESEARCH: A number of new strategies become very important in regenerative medicine. However, we discuss the limitations of stem cells and latest development in the reprogramming research.

Production of IL-10 and IL-12 by antigen-presenting cells in periapical lesions.

BACKGROUND: Interferon-γ (IFN-γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up-regulated by interleukin (IL)-12) and down-regulated by IL-10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. METHODS: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen-presenting cells was tested using a mixed leukocyte reaction. RESULTS: We observed the positive correlations between the levels of IL-12 and IFN-γ as well as IL-12 and IL-10 in cultures of mononuclear cells. As IL-10 and IL-12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14(+) adherent cells, produced high levels of IL-10 and very low levels of IL-12. In contrast, non-adherent, strongly HLA-DR(+) dendritic cells, potent stimulators of the alloreactive T-cell response, produced low levels of IL-10 and moderate levels of IL-12. Dendritic cells stimulated the production of IFN-γ by allogeneic CD4(+) T cells. In contrast, the level of IFN-γ was significantly decreased and the production of IL-10 was enhanced by addition of macrophages to the culture system. CONCLUSION: Our results suggest that a fine balance between the production of IL-10 and IL-12 by different antigen-presenting cells, through IFN-γ, may control the course of chronic inflammation in periapical lesions.

Metabolic syndrome attenuates ulcerative colitis: Correlation with interleukin-10 and galectin-3 expression.

BACKGROUND: Ulcerative colitis (UC) is a chronic disease characterized by inflammation of intestinal epithelium, primarily of the colon. An increasing prevalence of metabolic syndrome (MetS) in patients with UC has been documented recently. Still, there is no evidence that MetS alters the course of the UC. AIM: To test the influence of the MetS on the severity of UC and the local and systemic immune status. METHODS: Eighty nine patients with de novo histologically confirmed UC were divided in two groups, according to ATP III criteria: Group without MetS (no MetS) and group with MetS. RESULTS: Clinically and histologically milder disease with higher serum level of immunosuppressive cytokine interleukin-10 (IL-10) and fecal content of Galectin-3 (Gal-3) was observed in subjects with UC and MetS, compared to subjects suffering from UC only. This was accompanied with predomination of IL-10 over pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-17 (IL-17) in the sera as well as Gal-3 over TNF-α and IL-17 in feces of UC patients with MetS. Further, the patients with both conditions (UC and MetS) had higher percentage of IL-10 producing and Gal-3 expressing innate and acquired immune cells in lamina propria. CONCLUSION: Local dominance of Gal-3 and IL-10 over pro-inflammatory mediators in patients with MetS may present a mechanism for limiting the inflammatory process and subsequent tissue damage in UC.

Conformational analysis and in vitro immunomodulatory and insulinotropic properties of the frog skin host-defense peptide rhinophrynin-27 and selected analogs.

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1ß and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal ß-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10-6 M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies.

Correlation between phenotypic characteristics of mononuclear cells isolated from human periapical lesions and their in vitro production of Th1 and Th2 cytokines.

UNLABELLED: The development and progression of periapical dental lesions, mediated by the specific immune response, are poorly understood. In these processes, an interplay of different proinflammatory and immunoregulatory cytokines is of crucial importance. OBJECTIVES: To examine the activation of T helper 1 (Th1) and Th2 immune responses in 25 human periapical lesions based on the ex vivo production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by mononuclear cells (PL-MNC). METHODS: The levels of IFN-gamma and IL-4 in culture supernatants of PL-MNC, determined by ELISA, were correlated with concentrations of these cytokines in cultures of control MNC from peripheral blood (PB-MNC), cellular composition of inflammatory cells and phenotypic characteristics of PL-MNC. RESULTS: We detected high levels of IFN-gamma in all samples, after cell stimulation with phorbol myristate acetate and Ca(2+) ionophore, that were not statistically different from the levels of IFN-gamma in PB-MNC cultures. IL-4 was detected in 76% samples, but its concentrations were much lower than in PB-MNC samples. The levels of IFN-gamma were higher in cultures of PL-MNC isolated from periapical lesions with predominance of T cells (T-type lesions) and correlated positively with the proportion of antigen-presenting cells (macrophages and dendritic cells), CD4(+) T cells and IgG2(+) B cells/plasma cells. The levels of IL-4 correlated negatively with the proportion of macrophages, but positively with the number of mast cells and IgG4(+) cells. IL-18Ralpha, a stable marker of Th1 cells, was detected on a relatively small proportion of CD3(+) T cells and its expression correlated with the levels of IFN-gamma. However, the expression of ST2L, a stable Th2 cell marker, was not detected. The levels of Th1 and Th2 cytokines did not correlate with clinical characteristics of the lesions, defined by the presence of symptoms. CONCLUSION: Cumulatively, our results suggest the predominance of Th1 immune response in periapical lesions.

ST2 deletion increases inflammatory bone destruction in experimentally induced periapical lesions in mice.

INTRODUCTION: ST2 is a member of the interleukin (IL)-1 receptor family, and IL-33 is its natural ligand. ST2 signaling promotes Th2 immune response in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. The purpose of this study was to investigate whether ST2 gene deletion affects the development of experimentally induced periapical lesions in mice. METHODS: Pulps of mandibular molars from wild-type (WT) and ST2 knockout (ST2(-)/(-)) BALB/c mice were exposed and left open to the oral environment. After death, hemi-mandibles were isolated and prepared for histologic, immunohistochemical, and flow cytometric analysis. RESULTS: The expression of IL-33 and its receptor ST2 was higher in periapical lesions in WT mice compared with normal root apices (both P < .05). The increased periapical bone loss observed in ST2(-)/(-) mice was associated with enhanced influx of neutrophils, CD3+ CXCR3+ Th1 cells, and CD3+ CCR6+ Th17 cells and increased number of tartrate-resistant acid phosphatase+ osteoclasts (all P < .05). Furthermore, periapical lesions in ST2(-)/(-) mice contained increased percentages of T cells expressing interferon-γ, IL-17, tumor necrosis factor-α, and IL-6 (all P < .05). In comparison with WT mice, CD3+ receptor activator of nuclear factor kappa B ligand+ T cells were increased, whereas CD3+ osteoprotegerin+ T cells were decreased in the lesions of ST2(-)/(-) mice (both P < .05). CONCLUSIONS: ST2 deletion increases inflammatory bone loss in experimental periapical lesions in mice, which is associated with enhanced Th1/Th17 cell mediated periapical immune responses and increased osteoclastogenesis.

Cytotoxic effects of glass ionomer cements on human dental pulp stem cells correlate with fluoride release.

OBJECTIVES: Glass ionomer cements (GICs) are commonly used as restorative materials. Responses to GICs differ among cell types and it is therefore of importance to thoroughly investigate the influence of these restorative materials on pulp stem cells that are potential source for dental tissue regeneration. Eight biomaterials were tested: Fuji I, Fuji II, Fuji VIII, Fuji IX, Fuji Plus, Fuji Triage, Vitrebond and Composit. We compared their cytotoxic activity on human dental pulp stem cells (DPSC) and correlated this activity with the content of Fluoride, Aluminium and Strontium ions in their eluates. METHODS: Elution samples of biomaterials were prepared in sterile tissue culture medium and the medium was tested for toxicity by an assay of cell survival/proliferation (MTT test) and apoptosis (Annexin V FITC Detection Kit). Concentrations of Fluoride, Aluminium and Strontium ions were tested by appropriate methods in the same eluates. RESULTS: Cell survival ranged between 79.62% (Fuji Triage) to 1.5% (Fuji Plus) and most dead DPSCs were in the stage of late apoptosis. Fluoride release correlated with cytotoxicity of GICs, while Aluminium and Strontium ions, present in significant amount in eluates of tested GICs did not. SIGNIFICANCE: Fuji Plus, Vitrebond and Fuji VIII, which released fluoride in higher quantities than other GICs, were highly toxic to human DPSCs. Opposite, low levels of released fluoride correlated to low cytotoxic effect of Composit, Fuji I and Fuji Triage.

Proinflammatory and immunoregulatory mechanisms in periapical lesions.

Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high production of IL-17, positive correlation between IL-17 and IFN-gamma, but not between IL-17 and IL-23 production. Most IL-17(+) cells coexpressed IFN-gamma. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-gamma which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most B-type lesions higher levels of IL-5 and TGF-beta were observed, as well as positive correlation between the production of TGF-beta and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2 and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the production of IFN-gamma. IL-10 and TGF-beta inhibited the production of both Th1 and Th17 cytokines. Dendritic cells (DCs) from periapical lesions, composed of immature (CD83(-)), and mature (CD83(+)) myeloid type DCs and plasmacytoid (BDCA2(+)) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and TNF-alpha than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas the secretion of IFN-gamma was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1) Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2 and immunoregulatory cytokines are more significant for advanced types of lesions with the predominance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating inflammation.

[Comparative immunohistochemical and quantitative analysis of inflammatory cells in symptomatic and asymptomatic chronic periapical lesions].

BACKGROUND/AIM: It has been demonstrated that lymphocytes, plasma cells, macrophages and neutrophil granulocytes represent the predominant cells of the inflammatory lesion of the dental granulomas. Other cells, such as mast cells, eosinophils, dendritic cells comprise minor, but functionally important cell populations. Most of the data considering cells that take part in these processes have been derived from immunohistological studies. This study was undertaken with the aim to determine the phenotype profile of inflammatory cells of dental granulomas using immunohistochemical method in order to study the differences of their quantitative properties and distribution between symptomatic and asymptomatic lesions. METHODS: The material for the analysis originated from 42 individuals with clinic and radiographic diagnosis of chronic periapical lesions. The tissue was take either during the periradicular surgery, or tooth extraction. Cryostat tissue sections were stained using the alkaline phosphatase-antialkaline phosphatase assay (APAAP). This method is highly valid and sensitive using a panel of specific monoclonal antibodies: CD3, CD4, CD8, CD19, CD38, CD14, CD1a, CD83, CD80, CD86, CD45 and CD123. RESULTS: The composition of the cell population revealed that there was no homogenous and site-specific pattern of the distribution of inflammatory cells. The results of our investigation revealed that the majority of inflammatory cells comprised lymphocytes and plasma cells, followed by subpopulations CD4+, CD8+ and CD14+ cells. Much lower in number were CD80+, CD86+ and CD83+ and CD1a+ cells. There were no statistically significant differences in mean values of inflammatory cells number between symtomatic and asymptomatic lesions, with the exception of CD86+ cells, the number of which was statistically higher in symptomatic lesions. CONCLUSION: Inflamatory infiltrate cells in dental granulomas are dominated by T- and B-lymphocytes. It points out the complexity of immunopathogenic events in imitiating and progressing of dental granulomas that involve mechanisms of both cellular and humoral immunity. Regarding the quantitative presence of immunocompetent cells in symptomatic and asymptomatic lesions no statistically significant difference was determined unless in mature dendritic cells present in symptomatic lesions.

Interleukin-17 plays a role in exacerbation of inflammation within chronic periapical lesions.

Interleukin (IL)-17 plays an important role in inflammation and certain autoimmune diseases. However, its role in the pathogenesis of chronic dental periapical lesions has not been studied. Periapical lesion mononuclear cells (PL-MNC) were isolated from inflammatory cells and phenotypically analyzed by immunocytochemistry. The cells were cultured in vitro and IL-17 and IL-8 were measured in the culture supernatants. Controls were peripheral blood (PB) MNC. The level of IL-17 and the proportion of neutrophils were significantly higher in symptomatic lesions. In addition, the production of IL-17 was higher in culture supernatants of PL-MNC isolated from lesions with a predominance of T cells, and the IL-17 concentration correlated with the proportion of CD3+ and CD4+ cells. There was a positive correlation between the levels of IL-17 and IL-8 in the group of symptomatic lesions. The relationship between these cytokines was additionally confirmed on the basis of augmented production of IL-8 by both PL-MNC and PB-MNC treated with IL-17. Our results suggest that IL-17, by stimulating the production of IL-8, may play a role in exacerbating inflammation within chronic periapical lesions.