Immunoexpression of tumour necrosis factor-α, interleukin-1α and interleukin-10 on odontogenic cysts and tumours.

AIM: To analyse the immunoreactivity of IL-1α, TNF-α and IL-10 in odontogenic cysts and tumours and to investigate possible associations with established biological behaviours of these different lesions. METHODOLOGY: Immunohistochemical expression of anti-IL-1α, anti-TNF-α and anti-IL-10 antibodies was assessed on epithelium and mesenchyme of 20 radicular cysts (RCs), 20 residual cysts (RECs), 20 dentigerous cysts (DCs), 18 solid ameloblastomas (SAs), 20 keratocystic odontogenic tumours (KCOTs) and 15 dental follicles (DFs). Comparative analysis of data was performed using the nonparametric Wilcoxon signed-rank test and Kruskal-Wallis's test. RESULTS: Significantly greater expression of IL-1α in the epithelium was noted in RC, KCOT and SA (P = 0.01), whilst IL-10 and TNF-α was in the epithelium of RC, DC and KCOT (P < 0.01). In the mesenchyme, significantly greater immunopositivity was observed for IL-1α, IL-10 and TNF-α in KCOT, DC and RC (P < 0.01). In epithelial and mesenchymal tissues, there were a significant number of cases of RC and DC with IL-1α < IL-10 ratio (P < 0.01), whilst SA and KCOT showed IL-1α > IL-10 (P < 0.01). There was a significantly greater percentage of DF, DC and KCOT with TNF-α > IL10 ratio (P < 0.01). CONCLUSION: These results suggest involvement of the proteins in the pathogenesis of odontogenic cysts and tumours, with emphasis on the highest immunoreactivity of osteolysis stimulating factors in tumours with aggressive biological behaviour, such as SA and KCOT.

Metallothionein immunoexpression in non-syndromic and syndromic keratocystic odontogenic tumour.

BACKGROUND: To compare the metallothionein (MT) immunoexpression in non-syndromic and syndromic keratocystic odontogenic tumour (KOT), to correlate MT with cellular proliferation, and to evaluate the influence of inflammation in MT. STUDY DESIGN: Fourteen cases of KOT were submitted to immunohistochemistry for MT and Ki-67 analysis. The lesions were grouped according to their grade of inflammation, and statistical analysis was performed. RESULTS: MT was higher in non-syndromic KOT than in syndromic KOT (p<0.05). No statistical difference in Ki-67 could be identified; however, an inverse correlation was observed between MT and Ki-67 in both lesions. When analysing inflammation, non-syndromic KOT showed no differences in either MT or Ki-67. CONCLUSIONS: The MT immunophenotype of syndromic KOT was different from non-syndromic KOT. MT might not be involved in the proliferation control of both KOT. MT and Ki-67 immunoexpressions proved to be unaffected by inflammation in non-syndromic KOT.

[THE ROLE OF IMMUNOLOGICAL CHANGES IN ODONTOGENIC CYSTS].

The study involved 67 patients with odontogenic cysts (OC) aged 18 to 45 years, who were divided into groups: Group 1 (n = 67) patients with OC aged 18 to 45 years, group 2--control group, consisted of 20 healthy persons of similar age. We studied the characteristics of immune status and immunoreactivity in patients with odontogenic cysts. Condition of cellular and humoral immunity was assessed by using the methods of direct rosette developing with erythrocytes coated with monoclonal antibodies to CD3+, CD4+, CD8+, CD22+, CD4/CD8 indicators of immunoregulatory index and phagocytic immunity. State of nonspecific resistance was studied by determining the phagocytic activity of neutrophils and their oxygen dependent metabolism in NBT test. The concentration of cytokines (IL-6 and IL-4) in serum was determined by ELISA. During the study we found that in patients with (OC) developed significant changes in the structure of the immune response at the cellular as well as at the humoral level that makes it necessary to develop new individualized preventive measures along with existing therapies OC.

The immunoprofile of odontogenic keratocyst (keratocystic odontogenic tumor) that includes expression of PTCH, SMO, GLI-1 and bcl-2 is similar to ameloblastoma but different from odontogenic cysts.

BACKGROUND: The aggressive biological behavior of odontogenic keratocysts (OKCs), unlike that of other odontogenic cysts, has argued for its recent re-classification as a neoplasm, 'keratocystic odontogenic tumor'. Identification of mutations in the PTCH gene in some of the OKCs that were expected to produce truncated proteins, resulting in loss of control of the cell cycle, provided additional support for OKCs having a neoplastic nature. METHODS: We investigated the immunohistochemical expression of the sonic hedgehog (SHH) signaling pathway-related proteins, PTCH, smoothened (SMO) and GLI-1, and of the SHH-induced bcl-2 oncoprotein in a series of primary OKC (pOKC), recurrent OKC (rOKC) and nevoid basal cell carcinoma syndrome-associated OKCs (NBCCS-OKCs), and compared them to solid ameloblastomas (SAMs), unicystic ameloblastomas (UAMs), 'orthokeratinized' OKCs (oOKCs), dentigerous cysts (DCs) and radicular cysts (RCs). RESULTS: All studied lesions expressed the SHH pathway-related proteins in a similar pattern. The expression of bcl-2 in OKCs (pOKCs and NBCCS-OKCs) and SAMs was significantly higher than in oOKCs, DCs and RCs (P < 0.001). CONCLUSIONS: The present results of the immunoprofile of OKCs (that includes the expression of the SHH-related proteins and the SHH-induced bcl-2 oncoprotein) further support the notion of OKC having a neoplastic nature. As OKCs vary considerably in their biologic behavior, it is suggested that the quality and quantity of interactions between the SHH and other cell cycle regulatory pathways are likely to work synergistically to define the individual phenotype and corresponding biological behavior of this lesion.

Immunohistochemical analysis of the patterns of p53 and PCNA expression in odontogenic cystic lesions.

OBJECTIVE: the role of p53 expression in odontogenic lesions has not been fully determined, but has been associated with cell proliferation. The purpose of this study was to analyze p53 and proliferating cell nuclear antigen (PCNA) expression in 4 different odontogenic lesions. DESIGN: expression of p53 and PCNA was analyzed in radicular and dentigerous cysts, odontogenic keratocysts, and calcifying odontogenic cysts (Gorlin cysts) using monoclonal antibodies for detection of p53 and PCNA. RESULTS: PCNA expression was significantly greater in the basal layer of radicular cysts and in the suprabasal layer of odontogenic keratocysts; the percentage of p53 positive cells was significantly greater in the suprabasal layer of odontogenic keratocysts. CONCLUSIONS: The patterns of p53 and PCNA expression in dentigerous and radicular cysts were similar although the two lesions are of different origin. In odontogenic keratocysts and Gorlin cysts, results indicate a different pattern of tumor growth.

Lymphocyte­derived microparticles stimulate osteoclastogenesis by inducing RANKL in fibroblasts of odontogenic keratocysts.

Leukocyte­derived microparticles (LMPs) include neutrophil­, lymphocyte­ and monocyte­derived MPs. LMPs act as proinflammatory mediators in autoimmune diseases, infectious diseases and vascular diseases. The present study examined the hypothesis that the percentage of LMPs was increased in patients with inflamed odontogenic keratocysts (OKCs), and investigated the biological effects of Jurkat cell­derived MPs on the fibroblasts of OKCs in vitro. Cyst fluid MPs, obtained by centrifugation of samples from 20 patients with inflamed OKCs, 3 patients with uninflamed OKCs, 15 patients with radicular cysts (RCs) and 12 patients with inflamed dentigerous cysts (DCs), were analyzed by transmission electron microscopy, dynamic light scattering and immunofluorescence staining. The percentages and concentrations of cyst fluid LMPs were further determined by flow cytometry. The cytokine levels of apoptotic Jurkat cell­derived MPs and Jurkat cell supernatants were compared by cytokine antibody arrays. Fibroblasts were isolated from 3 patients with OKC and co­cultured with apoptotic Jurkat cell­derived MPs with or without interleukin (IL)­15Rα to detect the levels of matrix metallopeptidase 9 (MMP­9) and receptor activator of nuclear factor­κB ligand (RANKL) by reverse transcription­quantitative polymerase chain reaction and enzyme­linked immunosorbent assay. The supernatant from Jurkat MPs­treated fibroblasts was collected to make conditioned medium in which the osteoclastogenesis of Raw264.7 cells was determined. Antibodies against human soluble (s)RANKL were added to the conditioned medium to investigate the inhibitory effects. Mean percentages of lymphocyte­ and neutrophil­derived MPs were significantly higher in inflamed OKCs than in DCs. Significant elevations in IL­15 were detected in apoptotic Jurkat cell­derived MPs compared with that in Jurkat cell supernatant. Furthermore, higher levels of MMP­9 and RANKL were detected in Jurkat cell MP­treated OKC fibroblasts, and this was partially blocked by IL­15Rα. Increased osteoclast­like cell formation was observed in the Jurkat MPs­treated fibroblast supernatant and Raw264.7 co­culture groups. The anti­human sRANKL antibody in the Jurkat MPs­treated fibroblast supernatant group decreased the osteoclastogenesis of the Raw264.7 cells. These results indicate that LMPs serve as novel communication tools that contribute toward the bone resorption of inflamed OKCs by inducing RANKL of OKC fibroblasts via IL­15.

Immunoexpression of proteins involved in cytoskeleton remodeling in benign odontogenic lesions.

OBJECTIVE: The present study was designed to analyze the immunolocalization of proteins involved in cytoskeleton remodeling, such as moesin and Rho-A, in benign odontogenic lesions that present with expansive growth and invasive clinical behavior. MATERIALS AND METHODS: Expressions of moesin and Rho-A in odontogenic epithelium were evaluated by immunohistochemical analysis in 45 odontogenic lesions using monoclonal antibodies. RESULTS: Our results demonstrated strong membranous and cytoplasmic expressions of moesin in the epithelial cells in 66.7% and 44.4% of the odontogenic lesions, respectively. Furthermore, Rho-A expression in odontogenic epithelium was strong in the membrane and cytoplasm of 51.1% and 62.2% of the odontogenic lesions, respectively. A statistically significant correlation was found between the membranous and cytoplasmic expressions of moesin (p = 0.000) and those of Rho-A (p = 0.048) in odontogenic epithelial cells, while no statistically significant correlation was found between moesin and Rho-A expressions (p > 0.05). CONCLUSIONS: The present study confirmed the strong expressions of moesin and Rho-A by odontogenic epithelial cells, suggesting their involvement in the development of benign odontogenic lesions. However, this study has failed to detect the connection between the moesin and Rho-A interaction in expansive growth and local invasiveness of these lesions.

Effects of positive pressure in odontogenic keratocysts.

Intracystic fluid pressure is thought to be involved in odontogenic cyst growth. In this study, we investigated the effects of positive pressure on the expression of interleukin-1alpha (IL-1alpha), matrix metalloproteinases (MMPs), and prostaglandin E2 (PGE2) in odontogenic keratocysts to determine whether this pressure stimulates inflammatory cytokine production and signaling of osteoclastogenic events. Positive pressure enhanced the expression of IL-1alpha mRNA and protein in odontogenic keratocyst epithelial cells, and increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and the epithelial cells. The pressure-induced secretions were inhibited by an interleukin-1 receptor antagonist. Recombinant human interleukin-1alpha (rhIL-1alpha) increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in the fibroblasts. Furthermore, in the fibroblasts, rhIL-1alpha enhanced the expression of macrophage colony-stimulating factor (M-CSF) mRNA, and rhIL-1alpha-induced PGE2 increased the expression of nuclear factor kappaB ligand (RANKL) mRNA. Thus, positive pressure may play a crucial role in odontogenic keratocyst growth via stimulating the expression of IL-1alpha in epithelial cells.

Recognition of protein apparently specific to odontogenic keratocyst fluids.

Separate antisera were raised against keratocyst, dentigerous cyst, and radicular cyst fluids and used to analyse a range of fluids from cysts of known type. Samples were subjected to crossed immunoelectrophoresis into homologous antiserum through an intermediate gel containing antibody to whole human serum to screen out serum derived components. A major antigen, designated X, which seems to be of epithelial origin but is not a keratin, was identified in keratocyst fluids. X resolves as two bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 81 K and 89 K and its major antigenic epitope is associated with disulphide bonds. Of the cysts studied to date, antigen X has been found consistently and exclusively in fluids from keratocysts; its presence and detection is independent of total soluble protein concentration and thus offers real potential as a reliable marker for preoperative diagnosis.

Evidence for the presence of lactoferrin in odontogenic keratocyst fluids.

Investigations into the possibility that X (an antigen consistently present in aspirated odontogenic keratocysts, but not in most fluids from other cyst types), represented a keratinocyte component failed to identify the antigen as a keratin, involucrin, or one of the blood group substances. Antigen X was detected in human mixed and parotid saliva and in colostrum, as well as in a commercially obtained preparation of colostral IgA. The antigen was similar biochemically to both secretory component and lactoferrin but proved to be identical antigenically with lactoferrin. The origin of lactoferrin in keratocyst fluids remains uncertain, though the lining epithelium seems a more likely source than does the very variable, and often negligible, inflammatory infiltrate found in these lesions.

The aggressive nature of the odontogenic keratocyst: is it a benign cystic neoplasm? Part 1. Clinical and early experimental evidence of aggressive behaviour.

In this, the first of three articles on the aggressive nature of the odontogenic keratocyst (OKC), there is a review of clinical and histological observations which indicated that this was an aggressive lesion with a predilection for recurrence unlike the majority of other jaw cysts. This led to the tentative suggestion that the OKC might be a benign neoplasm. Subsequently there were early laboratory investigations that compared proliferation rates of the OKC epithelium with other jaw cysts, comparative enzyme histochemistry to assess aspects of its metabolism and markers that would enable accurate presurgical diagnosis of this cyst. Comparative studies were also pursued on the walls of the OKC and other jaw cysts to identify factors that might influence the capacity of the OKC to resorb the bone surrounding it. The clinical and laboratory studies reviewed in this section provided cogent presumptive evidence of the distinctively aggressive nature of the OKC that led numbers of investigators to pursue immunocytochemical and genetic studies on this cyst. Parts 2 and 3 of this series review this work.

Glandular odontogenic cyst in China: report of 12 cases and immunohistochemical study.

OBJECTIVE: The purpose of this study was to present 12 additional cases of glandular odontogenic cyst (GOC) in the Department of Oral Pathology, School of Stomatology, Wuhan University, People's Republic of China, and to investigate their immunohistochemical cytokeratins (CKs) expression in the epithelial components. METHODS: A total of 12 GOCs were reviewed clinically and radiographically, and immunohistologic CKs AE1, 7, 8/18, 10/13, 14, 16, 19 and 20 were performed by using a standard biotin-streptavidin immunoperoxidase technique on paraffin sections. RESULTS: The present series showed that eight occurred in males and four in females. The mean age was 37.6 years with a peak incidence occurring in the third decades (six of 12). Mandibles were more affected than maxillas (7:5), especially anterior mandible (four of seven). Radiographically, ratio multilocular to unilocular radiolucencies was 5:7 usually with well-defined borders. Histologically, cystic spaces were lined by non-keratinized stratified epithelia containing focal plaque-like or whirlpool-like thickenings; surface epithelial layer-containing eosinophilic cuboidal cells; mucous cells; and mucin pools of microcystic areas in the epithelium. Immunohistochemistry showed that epithelium of GOCs stained for CKs AE1, 7, 8/18, 10/13, 14 and 19 with slight changes in their patterns, and no reaction to CKs 16 and 20. CONCLUSIONS: Most clinical and histologic features in this study were analogous to those reported west population, although with slight difference between them. Histologically, the morphology of the epithelium strongly suggested an odontogenic origin, and CKs expression of GOC was similar to that of odontogenic epithelium, suggesting histochemically that GOC might be derived from odontogenic epithelium.

Demonstration and partial characterization of a novel soluble antigen present in keratocysts.

Keratocyst antigen was demonstrated in the fluid of keratinizing odontogenic cysts (primordial cysts) but not in fluids of other cyst types. The antigen was not present in plasma or saliva. It resisted collagenase digestion and did not react with keratin antibodies. In gel filtration, the antigen migrated as a single peak and gave a molecular weight of about 50 000 when analyzed in SDS-polyacrylamide gel electrophoresis. In immunoelectrophoresis, it migrated in the prealbumin region and was localized in epithelial cells of the cyst capsule, when studied by a double-antibody fluorescence technique. The origin and function of the keratocyst antigen is unclear but it may be a soluble marker for keratocyst differentiation.

Blood group antigens A and B in ameloblastomas, odontogenic keratocysts and non-keratinizing cysts.

The expression of blood group antigens A and B has been studied in 8 ameloblastomas, 16 odontogenic keratocysts from patients with basal cell nevus syndrome, 11 odontogenic keratocysts from patients without the syndrome, and 12 non-keratinizing odontogenic cysts, using a double layer immunofluorescence staining technique. The amount of antigen in the lesions was compared with the content of antigen in normal buccal mucosa from each patient. All ameloblastomas reacted negatively, three cysts from the patients with the basal cell nevus syndrome reacted negatively, and the odontogenic keratocysts from patients without the syndrome as well as the non-keratinizing odontogenic cysts all gave a positive reaction.

Epithelial cell markers and proliferating cells in odontogenic jaw cysts.

The expression of keratins, CEA, EMA, and rat liver antigen (RLA) and the presence of Ki67+ proliferating cells were studied in the epithelial linings of 50 odontogenic cysts using an indirect immunoperoxidase method on acetone-fixed frozen sections. All cysts were positive with monoclonal antibodies of broad keratin specificity (CK1, AE1-3), and between 40 and 100 per cent of epithelial cells expressed keratins 13 and 19. Keratins 7, 8, and 18 were rarely expressed although surface cells in areas of mucous metaplasia often expressed keratins 7 and 18. Expression of keratin 10/11 was related to the presence of a well-ordered epithelial lining and was detected in isolated cells in 4/32 non-keratinizing cysts and in the upper suprabasal cell layers of 17/18 keratocysts. Although CEA, EMA, and RLA were detected in the epithelium of all specimens, the pattern of expression of CEA and EMA differed between cyst types. Ki67+ proliferating cells were most prevalent in keratocyst epithelia, where they were usually found within lower suprabasal layers which were negative or weakly positive for keratins 10/11 and 13. These results indicate differences in keratin, CEA, and EMA expression between cyst types which appear to be dependent on epithelial differentiation/structure rather than cyst type or histogenesis. Although these differences may not be of diagnostic significance, the consistent expression of both keratins 13 and 19 may provide a useful marker of odontogenic epithelium in general.

[Immunopathologic mechanisms associated with granulomas and odontogenic cysts: a general review]. / Mécanismes immunopathologiques associés aux granulomes et kystes odontogènes: revue générale.

The authors make a general survey of the immunopathological mechanisms involved in odontogenic cysts and granulomas. The dental pulp and the sound periapical tissues contain neither inflammatory nor immunoglobin-producing cells. Dental decay associated with bacterial aggression of the dental pulp and periapical tissues might induce a nonspecific inflammation which subsequently becomes more specific. Immunoglobulins are present in fluid aspirates from odontogenic cysts and granulomas. Several investigations have pointed out nonspecific inflammatory mediators (including the C3 complement components), T lymphocytes (helpers and suppressors), B lymphocytes, protein S-100 + cells (Langerhans cells). The presence of immunoglobulins, immunocompetent cells and C3 complement components confirms that all constituents of both humoral and cell-mediated immunological reactions might play an essential role in the pathogenesis of osteolysis.

[Expression of certain cytokeratins in the epithelium of dentigerous and primordial cysts]. / L'expression de certaines cytokératines par l'épithélium de kystes dentigères et primordiaux.

The histopathologic diagnosis of odontogenic cysts is based mainly on the morphological nature of the epithelial lining of the cyst. A standard immunocytochemical method based on anticytokeratin monoclonal antibodies was used for the diagnosis of dentigerous and primordial cysts: 12 odontogenic cysts were diagnosed on clinical, radiological and pathological criteria in 9 dentigerous cysts and 3 primordial cysts. The anticytokeratin antibodies used in this study were KL1 (Immunotech, France) and AE1, AE2, AE3 and AE8 (ICN-Miles, France). The anticytokeratin antibodies used stained only the epithelial cells confirming their accuracy. The KL1 antibodies stained homogeneously the various epithelial cells. This positive reaction was not modified by the various fixation methods used. Some reactions observed with AE antibodies seemed to be modified by Bouin's fixative. The staining homogeneity of the primordial cysts and the staining heterogeneity of the dentigerous cysts seemed to be related to morphological aspects of their respective epithelia. The epithelial reactions in these 2 types of cysts towards inflammation were different.

Immunohistochemical detection of Fas antigen in oral epithelia.

A monospecific and high titer polyclonal antibody, designated as Fas D, raised against synthetic polypeptides selected from a part of the human Fas antigen (aa 104-114), was used to identify the Fas antigen in human oral epithelia in normal and pathological states. The human gingival proteins had been extracted and analysed by an ABC (avidin-biotin complex) immunoblotting technique. The antibody interacted with a single band in gingival proteins with an estimated molecular weight of 35,000, which is in good agreement with that calculated from amino acid sequences of the human Fas antigen. Using an indirect immunohistochemical method, the antibody localized on the stratum spinosum and the basal part of the stratum corneum of normal human gingiva. Specimens obtained from patients with odontogenic keratocysts, leukoplakia, lichen planus, and squamous cell carcinoma were also stained with the antibody. The pattern of the Fas antigen distribution in oral stratified squamous epithelia was, with some overlapping, characteristic for each disease.