Immunoglobulin producing cells in human periapical granulomas

Ten cases of periapical granuloma were stained for igG, IgA, IgM, IgD and IgE by the unlabelled antibody peroxidase-antiperoxidase complex method to investigate the class distribution of plasma cells within the lesions. The results indicated that IgG containing plasma cells predominated in all lesions and that IgG, IgA, IgM, IgE and IgD cells represented 81.9, 11.4, 5.4, 1.1 and 0.2 per cent respectively, of the total positive cell population. All specimens contained IgE plasma cells in addition to many non-lymphoid cells showing membrane-like staining for IgE. The results suggest that in addition to tissue damage via local deposition of immune complexes IgE mediated immediate hypersensitivity reactions may have a role in pathogenesis.     

Midkine expression in human periapical granulomas

Introduction

The expression of midkine (MK), a heparin-binding growth factor, is increased in various human tumors, making it a promising tumor marker and target for tumor therapy. MK is also related to the regulation of the development and etiology of chronic or autoimmune diseases; however, the involvement of MK in apical periodontitis has never been examined. This study compared the localization of MK-expressing cells and MK messenger RNA expression in periapical granulomas with healthy gingival tissues.

Methods

Periapical lesions were removed surgically from chronic apical periodontitis patients, and serial tissue sections were stained with hematoxylin-eosin. The lesions diagnosed as periapical granulomas pathologically were examined by immunohistochemistry using human MK monoclonal antibodies. MK messenger RNA expression was also detected using real-time polymerase chain reaction analysis. Healthy gingival tissues were analyzed in the same manner.

Results

MK was expressed by inflammatory cells, such as macrophages, lymphocytes, and neutrophils, as well as by endothelial cells in periapical granulomas but not in healthy gingival tissues. The MK-expressing inflammatory cells were seen adjacent to blood vessels, which contained MK-expressing endothelial cells, suggesting the interaction of MK among these cells during the process of inflammatory cell infiltration. Quantitative analysis of MK messenger RNA expression revealed that periapical granulomas expressed significantly more MK than healthy gingival tissues.

Conclusions

These findings suggest that MK is involved in the pathogenesis of periapical granulomas.     

The microbiology of periapical granulomas

Of the 16 periapical granulomas studied, 14 (88%) yielded a positive growth when homogenized and cultured. The concentration of colony-forming units per milliliter of the suspension ranged from 10(1.3) to 10(4.0) (mean 10(2.2)). A total of 47 isolates comprising 26 (55%) facultative anaerobes and 21 (45%) strict anaerobes were obtained. The organisms most commonly cultured were Veillonella species (15%), Streptococcus milleri (11%), Streptococcus sanguis (11%), Actinomyces naeslundii (11%), Propionibacterium acnes (11%), and Bacteroides species (10%). Most of the organisms (96%) were sensitive to either amoxicillin, clindamycin, or tetracycline, whereas only 45% were sensitive to metronidazole.     

Presence of CXCR3-positive cells and IFN-gamma-producing cells in human periapical granulomas

In an attempt to understand further the balance between the types of helper T (Th) cells in human apical periodontitis, we examined the difference in the expression of the chemokine receptor and cytokine in samples obtained from human subjects by means of immunohistochemical methods. Chemokine receptor CXCR3-positive cells and IFN-gamma-producing cells were found to be present in human periapical granulomas, whereas chemokine receptor CCR3-positive cells and IL-4-producing cells could not be detected. By contrast, no factor expression was observed in a clinically healthy periodontal ligament serving as a negative control. Our findings suggest that Th1 cells may play an important role in the pathological process of local inflammation such as apical periodontitis.     

Confocal immunolocalization of VE-cadherin- and CXC chemokine-expressing endothelial cells in periapical granulomas

Aim To determine whether endothelial cells (ECs) in periapical granulomas can express vascular endothelial (VE)‐cadherin, CXCL8 and CXCL10 by examining with two‐colour confocal laser scanning microscope. Methodology Periapical lesions were surgically removed from patients with chronic periapical periodontitis (n = 20), and the paraffin‐embedded sections were prepared after being fixed with cold acetone. The 7‐μm‐thick sections were stained with haematoxylin–eosin and then examined pathologically using a light microscope. The lesions diagnosed as periapical granulomas (17 specimens) were analysed further using immunofluorescence and antibodies specific for human VE‐cadherin, CXCL8, and CXCL10. The slides were carefully examined using a confocal laser scanning microscope. The numbers of positive ECs were counted, and the comparison between VE‐cadherin‐positive ECs and CXCL8 or CXCL10 was assessed statistically using one‐way ANOVA followed by a Student–Newman–Keuls test. Results The expression of CXCL8 and CXCL10 by ECs was detected in 60.4 ± 13.4 and 67.2 ± 13.9%, respectively. However, the percentage of VE‐cadherin‐expressing ECs was 40.4 ± 10.5%, which was significantly lower (P < 0.01) than CXCL8 and CXCL10‐expressing ECs. Two‐colour immunofluorescence staining revealed that ECs co‐expressed VE‐cadherin and CXCL8 (37.4 ± 14.1%) or CXCL10 (39.1 ± 13.8%). Conclusions VE‐cadherin expression in ECs was lower than CXCL8 and CXCL10, suggesting that inflamed ECs in periapical granulomas could increase vascular permeability and that leukocyte chemotaxis mediated by ECs might occur. These findings may suggest the possibility that ECs could play a pivotal role in cell recruitment in periapical granulomas.     

Interferon-γ-producing cells and inducible nitric oxide synthase-producing cells in periapical granulomas

Periapical granulomas contain a large number of T lymphocytes and monocytes/macrophages and a small number of B lymphocytes and polymorphonuclear leukocytes. Sections from eight periapical granulomas were stained by a variety of immunohistochemical methods. The vascular endothelial cells stained positively for intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Helper T cells were identified by immunostaining for CD4 and stained positively for interferon-γ (IFN-γ). However, CD4-positive T cells did not stain for interleukin-4 (IL-4). Monocytes/macrophages were identified by immunostaining for CD68 and stained positively for IL-lγ or inducible nitric oxide synthase (iNOS). IL-1β could not be detected in the same samples. No cytokine expression was observed in B cells identified by immunostaining for CD20. IFN-γ-and iNOS-positive cells could not be detected in clinically healthy periodontal ligament being used as a negative control. These results suggest that the IFN-γ-producing T cells and iNOS-positive cells may modulate the progress of disease in local inflammation sites such as in periapical granulomas.     

根尖周肉芽肿的扫描电镜研究

对１２例１４个患者有慢性根尖周炎伴根尖周肉芽肿形成的患牙进行根尖切除术和根尖肉芽肿刮治术。将切除的根尖和刮除的根尖周肉芽组织经处理后进行扫描电子显微镜观察，发现根尖孔内、外及根尖周肉芽组织内有大量的成堆的球菌。并发现有大量的炎性细胞浸润，炎性细胞以淋巴细胞主要是Ｔ淋巴细胞为主，有少量的巨噬细胞存在，说明根尖周肉芽肿的形成与根管内的感染有密切的关系。另外观察到根尖牙骨质的吸收以及上皮条索、纤维组织的存在推断牙周膜修复、上皮再附着的可能性     

Immunohistochemical analysis of FoxP3+ cells in periapical granulomas and radicular cysts

ObjectivesTo compare the number of FoxP3⁺ cells between periapical granulomas (PGs) and radicular cysts (RCs), and to correlate this number with the intensity of the inflammatory infiltrate in these lesions and with epithelial thickness of RCs.Study designThirty PGs and 30 RCs were submitted to immunohistochemical analysis using an anti-FoxP3 polyclonal antibody. FoxP3⁺ cells were counted under a light microscope (×400 magnification) in five fields and the mean value was calculated for each specimen. Statistical tests were used to evaluate differences in the number of FoxP3⁺ cells according to type of lesion (PG vs. RC), intensity of the inflammatory infiltrate (grade I/II vs. grade III), and epithelial thickness of RCs (atrophic vs. hyperplastic).ResultsFoxP3⁺ cells were detected in most PGs (93.3%) and RCs (93.3%). The median number of FoxP3⁺ cells was 2.40 in PGs and 1.00 in RCs, with this difference being statistically significant (P = 0.005). No significant differences in the number of FoxP3⁺ cells were observed in terms of the intensity of the inflammatory infiltrate (P = 0.465) or epithelial thickness of RCs (P = 0.737).ConclusionsThe present results suggest a greater participation of regulatory T cells in the modulation of the inflammatory response in PGs. In addition, the presence of a less effective regulatory environment in RCs, together with the high levels of inflammatory mediators as reported in the literature, may contribute to the greater growth potential of these lesions.     

Heparanase expression in periapical granulomas and radicular cysts

Heparanase is an endo-β-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.     

Radiographic features of periapical cysts and granulomas

Many studies have been reported on radiographic lesion sizes of periapical lesions. However no studies have been reported on prevalences of subjective radiographic features in these lesions except for the early assumption that a periapical cyst usually exhibit a radiopaque cortex. This study is conducted to evaluate the prevalences of several subjective radiographic features of periapical cysts and granulomas in the hope to identify features that maybe suggestive of either diagnosis. The results showed that a regular (circular or semi-circular) radiographic outline is likely to be a periapical cyst while an irregular radiographic outline is not indicative of either a cyst or a granuloma. The association between the regular/irregular radiographic outline and the type of periapical lesion was found to be statistically significant (p less than 0.001). The associations of two other radiographic features and the type of periapical lesion were found to be just statistically significant (p less than 0.01). These features are the symmetry/asymmetry of the radiolucency in relation to the apex and the funnel-shaped/angular-shaped extension of the radiolucency. The widely accepted criteria that the possession of a radiopaque cortex surrounding the radiolucency can discriminate between a periapical cyst and granuloma cannot be statistically supported in this study.     

Examination on Candida spp. in refractory periapical granulomas

Aim To examine the occurrence of Candida spp. in refractory periapical granulomas. Methodology One hundred and three surgically removed periapical granulomas were subjected to molecular analysis for the occurrence of Candida albicans. DNA was extracted from the samples using a modified phenol/chloroform/isoamyl alcohol method and was subjected to polymerase chain reaction (PCR) with OPA‐03 and repetitive sequence (GACA)₄ primers. The PCR products were separated in agarose gel electrophoresis, stained with ethidium bromide, visualized using UV light and the sequences were analysed. Samples indicating possible occurrence of Candida were further investigated by histological and immunohistological methods. Periodic acid–Shiff staining (PAS) was used to detect yeast cells and hyphae, and specific monoclonal antibodies to recognize high molecular mass mannoproteins present in the C. albicans cell wall. DNA extraction was controlled by running PCR using β‐actin primers (a housekeeping gene). C. albicans CCUG19915, C. tropicalis ATCC750, C. krusei ATCC6258, C. guilliermondii ATCC6260 and C. glabrata CCUG32725 served as positive controls in PCR. A tissue preparation of chronic atrophic candidosis in oral buccal mucosa served as a positive control for histological and immunohistological examinations. Results Polymerase chain reaction with β‐actin primers indicated successful DNA extraction in 68 out of 103 samples. The majority of the samples (50) were negative whereas 18 of the samples showed PCR products indicating possible occurrence of Candida spp. PAS‐staining and immunohistological examination of these samples were, however, negative. Further analysis of the PCR products revealed sequences not typical for Candida spp. Conclusions Candida spp. do not seem to occur in periapical granuloma.     

Expression of regulatory apoptotic proteins in peripheral giant cell granulomas and lesions containing osteoclast-like giant cells

Peripheral giant cell granuloma consists of monononuclear cells and osteoclast-like giant cells. The proliferative ability of peripheral giant cell granuloma is restricted to the mononuclear cell compartment, whereas multinucleated giant cells lack mitotic activity. Although the proliferative compartment of peripheral giant cell granuloma has been investigated in detail, the expression and distribution of proteins regulating apoptosis is unknown. The present study demonstrates strong expression of bak and bax in the majority of giant cells. In contrast, giant cells show only weak positivity for bcl-2 and moderate positivity for bcl-x. Mononuclear cells were negative to weakly positive for bcl-x. Only scattered mononuclear cells were positive for bak, bax and bcl-2. The frequency of apoptotic nuclei detected by TUNEL-staining compared to regular nuclei was 18 times higher in giant cells than in mononuclear cells. In summary, our findings support the presumption that giant cells of bone and soft tissue tumors are reactive cell forms and not of neoplastic origin.     

Cytotoxic T lymphocytes versus streptococcal colonization in periapical granulomas.

Seventeen dental periapical lesions were investigated to study bacterial colonization. Periapical lesions, obtained after apicotomy, were also enzymatically desegregated to quantitatively analyze lymphocyte subpopulations by flow cytometry. Fourteen samples yielded a positive bacterial growth when homogenized and cultured. We isolated enough lymphocytes to make flow cytometric analysis from 12 samples. A significant increase in interleukin-2 receptor and ICAM-1 molecule expression on T cells was found, compared with peripheral blood lymphocytes. Furthermore, a decreased expression of interleukin-2 receptors and HLA DR molecules on CD8+ T cells was found in granulomas predominantly colonized by Streptococcus spp., compared with lesions predominantly colonized by strict anaerobes.     

Langerhans' cell histiocytosis in association with periapical granulomas and cysts.

This report describes a series of six cases of inflammatory periapical disease with small aggregates of Langerhans cells as a minor component. Immunohistochemical findings confirm that the cells are phenotypically related to Langerhans cells. Aggregates of these cells are not normally found in radicular cysts or periapical granulomas and have been interpreted to represent chronic localized Langerhans' cells histiocytosis. Whether these lesions, which arise within the context of chronic inflammatory periapical disease, represent incipient eosinophilic granulomas or are a more benign, minimally destructive form of Langerhans' cell histiocytosis is unknown. Clinical follow-up suggests that these lesions remain localized and that curettage is adequate treatment.     

Comparative immunohistochemical study of the presence of mast cells in apical granulomas and periapical cysts: possible role of mast cells in the course of human periapical lesions

Cells other than macrophages and lymphocytes have recently been shown capable of producing cytokines and mediators. Among these are mast cells, a cell population now recognized for its immunoregulatory properties. Little is known about the complex interactions between cells, cytokines, and other inflammatory elements in periapical lesions. The objective of this investigation was to determine the immunohistochemical pattern of expression of mast cells tryptase in periapical lesions based on study of 20 apical granulomas and 20 periapical cysts. Microscopic analysis revealed mast cells to be present in greater numbers in periapical cysts than in apical granulomas, and in cysts were more numerous in regions of active inflammation. Mast cells tended to be more common in the peripheral regions of both periapical lesions, and were often found in close proximity to lymphocytes. These findings lead us to propose a functional relationship between these two cell populations that may facilitate elicitation of an immune response contributory to the pathogenesis of periapical lesions.