The expression of TNF-α in recurrent aphthous stomatitis: A systematic review and meta-analysis.

OBJECTIVE: The pathogenesis of recurrent aphthous stomatitis (RAS) is related to an increase of pro-inflammatory cytokine, namely tumor necrosis factor α (TNF-α). This cytokine plays an important role in the development of ulcer lesions, both in saliva, tissues and blood. This systematic review analyzed the differences of TNF-α in lesions, salivary and blood and can be used as a reliable method of diagnosis for RAS. METHODS: A comprehensive search of PubMed, Scopus databases, Web of Science, Scielo, Google Scholar and Embase with keywords. The inclusion criteria were studies that assessed the saliva, serum, and RAS lesion, with the outcome reporting the mean of saliva, serum and tissue expression of TNF-α. The risk of bias was also assessed. RESULT: Healthy individuals showed significantly lower TNF-α than RAS (SMD = -1.517, 95% CI [-2.25, -0.78]). Although there is a significant difference between sample (i.e., saliva, serum) and detection type (i.e., cytometry bead array, ELISA), both methods can detect a significant difference in TNF-α between healthy individuals and RAS patients. CONCLUSIONS: The TNF-α is a useful diagnostic marker for RAS. We encourage saliva to detect changes in TNF-α during ulceration as it provides accuracy, reliability, and non-invasive procedure compared to a blood draw.

Recurrent, Severe Aphthous Stomatitis and Mucosal Ulcers as Primary Manifestations of a Novel STAT1 Gain-of-Function Mutation.

Chronic mucocutaneous candidiasis (CMC) characterized by persistent and recurrent Candida infection of the skin, nails, and the mucosa membranes has been proposed as the major infectious phenotype in patients with gain-of-function mutation of signal transducer and activator of transcription 1 (STAT1) 1. However, viral infections caused mostly by herpesviruses, and a broad range of autoimmune disorders may also be part of the clinical phenotype. We report here on a 31 years old female patient suffering from severe mucosal aphthous mucositis and ulcers and recurrent herpes simplex for decades. We found a previously unknown heterozygous sequence variant in STAT1 (c.1219C>G; L407V) affecting the DNA-binding domain of the protein in the patient and her 4 years old daughter. We found this mutation gain-of-function (GOF) by using immunoblot and luciferase assays. We detected low proportion of IL-17A-producing CD4+ T cell lymphocytes by using intracellular staining and flow cytometry. Candida-induced secretion of IL-17A and IL-22 by mononuclear cells from the patient was markedly decreased compared to controls. These data suggest that the novel mutant allele may result in impaired differentiation of CD4+ T cells to CD4+/IL-17+ cells. The clinical phenotype of the disease in this patient was unique as it was dominated primarily by severe aphthous stomatitis and ulcerative esophagitis and only partly by typical CMC resulting in diagnostic delay. We suggest that patients with severe recurrent aphthous stomatitis and esophagitis should be evaluated for STAT1 GOF mutation. Based on the broad clinical spectrum of the disease, we also suggest that CMC and CMC disease may not be an appropriate term to define clinically STAT1 GOF mutation.

LncRNA CASC 2 is upregulated in aphthous stomatitis and predicts the recurrence.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is a common oral disease with unknown molecular pathogenesis. Our preliminary microarray analysis revealed the altered expression of lncRNA Cancer Susceptibility Gene 2 (CASC2) in RAS. We therefore analyzed the role of CASC2 in RAS. METHODS: In this study, plasma samples were obtained from RAS patients and healthy participants. Plasma levels of CASC2 were measured by RT-qPCR. Plasma levels of IL-6 and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA). A follow-up study was performed to analyze the role of CASC2 in the recurrence of RAS. RESULTS: In the present study, we found that lncRNA Cancer Susceptibility Gene 2 (CASC2), as well as pro-inflammatory factors interleukin 6 (IL-6) and interleukin 18 (IL-18), were upregulated in plasma of RAS patients compared with healthy participants. Plasma levels of lncRNA CASC2 were positively correlated with plasma levels of IL-6 and IL-18 in RAS patients but not in healthy participants. Compared with pre-treatment levels, plasma levels of lncRNA CASC2, IL-6 and IL-18 were reduced after recovery. A follow-up study showed that patients with high levels of lncRNA CASC2 had a significantly higher recurrence rate. CONCLUSION: LncRNA CASC 2 is upregulated in RAS and predicts the recurrence.

Basic Characteristics of Adults with Periodic Fever, Aphthous Stomatitis, Pharyngitis, and Adenopathy Syndrome in Comparison with the Typical Pediatric Expression of Disease.

Autoinflammatory diseases are caused by inflammasome dysregulation leading to overproduction of proinflammatory cytokines and a pathological delay in the inflammation switching off. The progress of cellular biology has partially clarified pathogenic mechanisms behind monogenic autoinflammatory diseases, whereas little is known about the polygenic ones. Although the genetic susceptibility of periodic fever, aphthous stomatitis, pharyngitis, and adenopathy (PFAPA) syndrome is still obscure, the presence of overlapping symptoms with monogenic periodic fevers, the recurrence in family members, the important role played by dysregulated interleukin- (IL-) 1ß secretion during flares, the overexpression of inflammasome-associated genes during attacks, and, last but not least, the therapeutic efficacy of IL-1ß blockade strongly indicate a potential genetic involvement in its pathogenesis, probably linked with environmental factors. PFAPA syndrome has a typical inception in the pediatric age, but a delayed onset during adulthood has been described as well. Treatments required as well as effectiveness of tonsillectomy remain controversial, even if the disease seems to have a self-limited course mostly in children. The purpose of this review is to provide an overview of this complex polygenic/multifactorial autoinflammatory disorder in which the innate immune system undoubtedly plays a basic role.

Increased beta 2 defensin in recurrent aphthous ulcer.

OBJECTIVES: It was hypothesized that beta 2 defensin (BD-2) is increased in RAU lesions compared with healthy controls to promote anti-microbial host defence. METHODS: RAU and control mucosa samples were subjected to quantitative real-time PCR and immunostained for BD-2, CD68, mast cell tryptase and 4-hydroxynonenal (4HNE). The effect of tumour necrosis factor-α (TNF-α) ± interleukin-17C (IL-17C), without and with vitamin K3, was studied on BD-2 expression in epithelial SCC-25 cells. RESULTS: Although BD-2 mRNA did not differ between healthy and RAU mucosa, BD-2 stained strongly in acute-phase RAU epithelium (P = 0.001). In controls, subepithelial BD-2(+) cells were mast cells and macrophages, whereas in RAU, most infiltrating leucocytes were BD-2(+) (P = 0.004). In cell culture, BD-2 was increased 124-fold by TNF-α (P < 0.0001) and 208-fold synergistically together with IL-17C (P < 0.0001). 4HNE staining of RAU epithelium was not significantly increased, and vitamin K3-induced reactive oxygen species (ROS) did not affect BD-2. CONCLUSIONS: Anti-microbial BD-2 was not affected by oxidative stress but was highly increased in the epithelial and immigrant cells in the acute-phase RAU lesions, probably in part synergistically by TNF-α and epithelial IL-17C, which are known to be induced by activation of danger-signal receptors by pathogen- and/or damage-associated molecular patterns.

Evaluation of hbeta;D-1 and hbeta; D-2 levels in saliva of patients with oral mucosal diseases / Evaluación de los niveles de hbeta; D-1 y hbeta;D-2 en la Saliva de pacientes con enfermedades de la mucosa oral

OBJECTIVE: This study aimed to determine a possible correlation between oral mucosal disease and salivary concentrations of the antimicrobial peptides human beta-defensin-1 (hβD-1) and human betadefensin- 2 (hβD-2). METHOD: The present work focussed on the establishment of a reversed phase-high performance liquid chromatography (RP-HPLC) procedure to quantify human beta-defensins (hβD-1 and hβD-2) in saliva samples of patients with oral diseases such as lichen planus (n = 10), Behçet (n = 10) and recurrent apthous stomatitis (n = 10). RESULTS: Linear calibration range for hβD-1 and hβD-2 defensins was 1.67−200 µg mL-1 and 3.13− 100 µg mL-1 with R2 values of 0.9998 and 0.996, correspondingly. The concentration of beta-defensins in saliva was determined by comparing the peak areas of eluted hβD-1 and hβD-2 with that of their standards. The variation of the amount of beta-defensins was evaluated by comparisons of the results obtained from the patients with oral mucosal diseases before and after treatments and the control subjects. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.62 µg mL- 1 and 5.39 µg mL-1 for hβD-1 and 0.94 µg mL-1 and 3.13 µg mL-1 for hβD-2, respectively. CONCLUSION: The salivary beta-defensin concentration was significantly higher in patients with oral mucosal diseases than in healthy volunteers; furthermore, in patients with oral mucosal diseases, the concentration was significantly higher before treatment than after treatment.

OBJETIVO: Este estudio tuvo por objeto determinar una posible correlación entre la enfermedad de la mucosa oral y las concentraciones salivales de la beta-defensina humana 1 (hβD-1) y la beta-defensina humana 2 (hβD-2) de los péptidos antimicrobianos. MÉTODO: El presente trabajo estuvo encaminado al establecimiento de un procedimiento de cromatografía líquida de alta eficacia de fase reversa (RP-HPLC) para cuantificar las beta-defensinas humanas (hβD-1 y hβD-2) en muestras de saliva de pacientes con enfermedades orales como el liquen plano (n = 10), Behçet (n = 10), y la estomatitis aftosa recurrente (n = 10). RESULTADOS: El rango de calibración lineal de las defensinas hβD-1 y hβD-2 fue 1.67-200 µg mL-1 y 3.13-100 µg mL-1 con valores R2 de 0.9998 y 996, respectivamente. La concentración de beta-defensinas en la saliva se determinó utilizando el área de sus estándares. La variación de la cantidad de beta defensinas fue evaluada por comparaciones de los resultados obtenidos de los pacientes con enfermedades de la mucosa oral, antes y después de los tratamientos y los sujetos de control. Se halló que el límite de detección (LDD) y el límite de cuantificación (LDC) fueron 1.62 µg mL-1 y 5.39 µg mL- 1 para hβD-1 y 0.94 µg mL-1 y 3.13 µg mL-1 hβD-2, respectivamente. CONCLUSIÓN: La concentración de beta-defensina salival fue significativamente mayor en los pacientes con enfermedades de la mucosa oral que en los voluntarios sanos. Además, en pacientes con enfermedades de la mucosa oral, la concentración fue significativamente mayor antes del tratamiento que después del tratamiento.

Characterization of lymphoid follicles with red ring signs as first manifestation of early Crohn's disease by conventional histopathology and confocal laser endomicroscopy.

BACKGROUND AND AIMS: Clinical observations suggest that the lymphoid follicles (LFs) may play a crucial role in the pathogenesis of inflammatory bowel disease (IBD), especially in Crohn's disease (CD) as the site of initial mucosal inflammation. The aim of this study was to compare the morphology of LFs in CD, ulcerative colitis (UC) and control patients using confocal laser endomicroscopy (CLE) in correlation to histological and immunohistochemical findings of biopsies. METHODS: 79 patients with IBD (46 with CD, 32 with UC and 1 patient with indeterminate colitis) and 67 controls patients were enrolled prospectively in this study. Median age was 32.5 years (range 19-65) and 37.4 years (range 20-65 years) respectively. To analyze the LFs, standardized images from the terminal ileum and the colon were taken using white-light video endoscopes. Additionally, CLE was performed to analyze subsurface structure of LFs. Targeted biopsies of LFs were analyzed using haematoxylin and eosin stain and immunohistochemistry. RESULTS: LFs were seen in all parts of the lower GI tract, but mostly in the terminal ileum and cecum. Endoscopy in 15 out of 17 patients with the first manifestation of CD showed LFs surrounded by red ring (so-called red ring sign, RRS). Histologically, LFs with RRS showed hypervascularization at the base of the LFs associated with numerous CD15-positive granulocytes. Similar features were not seen in LFs without RRS and in the control group. In some LFs with RRS early aphthous ulcers were seen. Using CLE, RRS showed abolished normal crypt architecture, crypt distortion, increased cellular infiltrate within the lamina propria, and dilated vessels. CONCLUSION: LFs with RRS probably represent an early sign of aphthous ulcers in early CD and, thus, may be considered as early markers of first manifestation and flares in CD.

Expression of apoptosis regulatory proteins p53, bcl-2 and bax in recurrent aphthous ulceration.

BACKGROUND: Recurrent aphthous ulceration (RAU) is considered to be an acute inflammatory disease of unknown pathogenesis. Apoptosis may represent an important event in the control of inflammation. OBJECTIVES: The aim of this study was to investigate apoptosis process in RAU using immunohistochemistry. METHODS: We studied the expression and location of p53, bcl-2 and bax in ulcerated lesions clinically diagnosed as RAU (n = 12) and compared it with that of oral clinically normal mucosa (n = 6) and of other inflammatory chronic disease such as oral fibrous inflammatory hyperplasia (FIH; n = 18). RESULTS: Significant statistically differences (n < 0.05) in p53 expression were noticed in RAU when compared with normal mucosa. No significant statistically differences (P > 0.05) were noticed between FIH and RAU. Bcl-2 and bax did not show remarkable differences between groups. CONCLUSIONS: Taken together, the data suggest that RAU induces p53 immunoexpression. Therefore, the protein might be related to the aetiopathogenesis of the ulcerated oral lesions.

HSP 60 expression in recurrent oral ulcerations of Behçet's disease.

OBJECTIVE: The aim was to investigate heat shock protein 60 (HSP60) expression in oral ulcerations of Behçet's disease (BD). STUDY DESIGN: Biopsy specimens were obtained from patients with BD (n = 11), recurrent aphthous stomatitis (RAS) (n = 11), oral lichen planus (OLP) (n = 11) and healthy individuals (HI) (n = 11). Eight samples in BD and RAS groups were evaluable. All groups were analyzed by biotin streptavidin-aminoethylcarbazole using monoclonal mouse antibodies to HSP60 Ab-1 (clone LK1). RESULTS: Immunostaining indicative of HSP60 expression in BD and RAS were significantly higher than HI. No significant difference was found between BD and OLP except in the suprabasal layer of epithelium. CONCLUSIONS: Altered expression of HSP60 was found in ulcerative lesions of BD and RAS suggesting that HSP60 has an association with the etiology or chronicity of these inflammatory lesions.

Rapid cell senescence and apoptosis in lymphocytes and granulocytes and absence of GM-CSF receptor in congenital dysgranulopoietic neutropenia.

A girl with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother with aphthae (A) were investigated. Apoptosis in lymphocytes and granulocytes of both patients (mother A+) were documented by high annexin and electron microscopic morphology. CD11b/CD18 of the daughter's granulocytes ranged between low to normal while that of the mother changed between very low to high levels through A(-) to A(+) periods. In both patients, CD11b/CD18 on lymphocytes were high; GM-CSF receptor was negative; CD4-/CD8- lymphocytes were high and the leukocytes which showed abnormal cell cycle were stained by senescence associated beta-galactosidase. We think that increased apoptosis and rapid cell senescence of leukocytes underlies the pathophysiology of CDN.

Association between recurrent aphthous stomatitis and salivary thiocyanate levels.

The aim of this work was to investigate the association between recurrent aphthous stomatitis (RAS) and salivary thiocyanate levels. The sample comprised men and women of age ranging from 15 to 55 years, who were allocated to four groups: 28 patients in RAS active phase (group 1); 28 patients in RAS remission phase (group 2); 29 smokers (group 3); 26 non-smokers without RAS (group 4). Samples of whole saliva mechanically stimulated were collected, and thiocyanate levels were measured. The results were analyzed by ANOVA and paired t-test. Mean salivary thiocyanate values were 0.55 mM, 0.64 mM, 2.36 mM and 0.96 mM in groups 1 (active RAS), 2 (remission RAS), 3 (smokers) and 4 (control), respectively. There was no significant difference in thiocyanate levels when groups 1 and 2 were compared with group 4. Group 3 showed a significantly higher thiocyanate concentration when compared with groups 1, 2 and 4 (P < 0.05). There was no significant difference in thiocyanate levels between groups 1 and 2 (P > 0.05). It is therefore suggested that there is no association between RAS and salivary thiocyanate levels.

Salivary interleukin-6 and tumor necrosis factor-alpha in patients with recurrent aphthous ulceration.

BACKGROUND: Recurrent apthous ulceration (RAU) is a well known oral disease which seems to be mediated principally by the immune system. However, it is still a matter of debate which part of the immune system is implicated in its pathogenesis as a reaction to the still unknown antigen. The aim of this study was to evaluate salivary cytokines, interleukin (IL)-6, and tumor necrosis factor (TNF)-alpha. METHODS: In 26 patients with minor RAU, age range of 23-49 years (mean 27.3 years), during both the acute phase and remission and in 26 healthy controls, age range of 22-64 years (mean 30.1 years), salivary IL-6 and TNF-alpha levels were determined by use of enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed by use of descriptive statistics. RESULTS: Significant differences in salivary TNF-alpha between healthy controls and patients with acute RAU and during the remission period were found (P < 0.001) as well as between patients with acute RAU and those during the remission period (P < 0.001). No differences in salivary IL-6 between all three groups could be found. CONCLUSIONS: We might conclude that elevated salivary TNF-alpha levels during acute RAU and especially during the remission period are of importance in RAU, whereas salivary IL-6 levels seem not to play a role in the RAU disease.

Expression of cytokines, chemokines, and chemokine receptors in oral ulcers of patients with Behcet's disease (BD) and recurrent aphthous stomatitis is Th1-associated, although Th2-association is also observed in patients with BD.

OBJECTIVE: Although the pathogenesis of Behcet's disease (BD) is unknown, immune dysfunction appears to be involved. To improve understanding of the role of T cells and cytokines in BD, the current study analysed the localization and extent of expression of T cell subsets, cytokines, chemokines, and chemokine receptors in oral ulcers from BD patients and for comparison in oral ulcers from patients with recurrent aphthous stomatitis (RAS), as well as in healthy oral mucosa. METHODS: Biopsies from oral ulcers of 25 BD patients and 19 RAS patients and oral mucosa from six healthy volunteers were immunoperoxidase stained. RESULTS: Both CD4- and CD8-positive T cells were present in the oral ulcers of BD and RAS patients. The T helper (Th)1 cytokines interleukin (IL)-12, interferon (IFN)-gamma, and tumour necrosis factor (TNF)alpha and the Th1-associated chemokine receptors CCR5 and CXCR3 were increased in both patient groups as compared to normal controls, indicating the involvement of a Th1 immune response in the immunopathology of both BD and RAS. However, the Th2 cytokine IL-4 was only observed in oral ulcers of BD patients but not in RAS patients. CONCLUSION: This is the first study that shows the presence of pro-inflammatory cytokines, as well as Th1-associated chemokine receptors, in the oral ulcers of BD patients, as well as RAS patients, at a protein level. However, the expression of the Th2 cytokine IL-4 within the oral lesions of only BD patients is suggestive of a more complex antigenic stimuli in BD patients compared with RAS patients.

Expressions of vascular endothelial growth factor and CD34 in oral aphthous lesions of Behçet's disease.

OBJECTIVE: Behçet's disease (BD) is an immunoin-flammatory vasculitis with an unknown etiopathogenesis. Vascular endothelial growth factor (VEGF) is a cytokine-stimulating angiogenesis. It has been suggested to play a role in inflammation and pathogenesis of vasculitic processes. STUDY DESIGN: VEGF and CD34 expressions were assessed in samples taken from oral aphthous lesions. The patients were evaluated for disease activity, duration of lesions, serum C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR). RESULTS: Twenty-six patients were included. Fourteen (53.9%) had increased CRP levels and 12 (46.1%) had increased ESR levels. Positive VEGF and CD34 staining were detected in 46.2% and 69.2% of biopsy samples, respectively. There was good positive correlation between them. The frequency of positive VEGF and CD34 staining was statistically higher in lesions with a duration of more than 6 days. No correlation was found between positive VEGF staining and serum CRP level, ESR and disease activity. CONCLUSION: Increased VEGF expression in correlation with CD34 positivity in oral aphthous lesions may show the role of VEGF in pathogenesis of these vasculitic lesions. We have concluded that VEGF may play a role during the course of oral aphthous lesions in BD.

The levels of plasma and salivary antioxidants in the patient with recurrent aphthous stomatitis.

BACKGROUND: Despite plenty of research, the cause of recurrent aphthous stomatitis (RAS) remains obscure. It has been proposed that, the aetiological factors such as local trauma, smoking, vitamin deficiencies and viral infections lead to aphthae formation via final common pathway based on increased oxidative stress. The aim of this investigation was to evaluate the antioxidant enzyme superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) alterations in plasma and saliva, and in addition uric acid (UA) in saliva, in patients with RAS and healthy controls. METHODS: Thirty-two patients with RAS and 30 healthy controls were included into the study. The SOD, CAT, GSHPx and UA levels were measured in plasma and saliva in study and control groups. RESULTS: In the RAS group, although the mean SOD (P<0.001) and CAT (P<0.05) levels of plasma were lower, GSHPx (P<0.001) levels were higher than control group. The salivary concentrations of the SOD (P<0.001), CAT (P<0.05) and GSHPx (P<0.001) in RAS group were entirely opposite to plasma concentrations. UA were not significant between RAS group and controls. CONCLUSION: Since we found salivary SOD and CAT levels were high whereas plasma levels were low, it has been thought that, salivary defence mechanisms via antioxidant agents may be stimulated against to the ulcerous lesion. We consider that the organism might mobilize the antioxidant potential to the sites where they were needed. At this point, decrease of SOD and CAT levels in the plasma may be related to this shift. It is also thought that GSHPx secretion in the saliva may also be increased but the increase in its turnover may be responsible for the diminished activity.

High-level expression of vascular endothelial growth factor and its receptors in an aphthous ulcer.

BACKGROUND: Aphthous ulcers are an extremely common disorder of unknown etiology. These ulcers cause significant morbidity through pain and interference with eating. Thalidomide, an angiogenesis inhibitor, is efficacious for the treatment of aphthous ulcers. METHODS: In situ hybridization was performed on an idiopathic aphthous ulcer using probes specific for the angiogenesis factor vascular endothelial growth factor, and its receptors, in order to determine whether these ulcers are highly angiogenic. CONCLUSIONS: Aphthous ulcers are highly angiogenic. Thalidomide may act to heal aphthous ulcers by inhibiting angiogenesis and promoting reepithelialization. Excess angiogenesis may inhibit reepithelialization in certain types of ulcers, and angiogenesis inhibitors may actually promote wound healing if ulcers are caused by excess angiogenesis.

Salivary levels of vascular endothelial growth factor (VEGF) in recurrent aphthous ulceration.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic cytokine involved in angiogenesis and wound healing. Its presence in recurrent aphthous ulceration has not been reported to date. The aim of this study was to investigate the association of salivary levels of VEGF with various stages of recurrent aphthous ulceration (RAU). METHODS: VEGF levels were determined in a group of 27 age and sex-matched healthy controls and in 30 patients with minor and major RAU grouped into the three stages: (I) early active stage, (II) active stage, and (III) remission period. VEGF levels (pg/ml; mean +/- SD) in unstimulated whole saliva were determined by enzyme immunoassay. RESULTS: Patients with major RAU - stages I and II - had decreased VEGF values (765 +/- 458 and 341 +/- 109, respectively) when compared both to healthy controls (1652 +/- 567; P < 0.01) and to stage III major RAU (1524 +/- 784; P < 0.005). CONCLUSION: Salivary VEGF levels seemed to be associated with ulcer development in major RAU, showing stage-dependent alterations during the course of this disorder.

Immunolocalization of tumor necrosis factor-alpha expressing cells in recurrent aphthous ulcer lesions (RAU).

Tumor necrosis factor (TNF)-alpha is a pro-inflammatory cytokine and crucial mediator in many aspects of immunity. Although several studies have shown that recurrent aphthous ulcers (RAU) can be prevented by treatment that prevents the synthesis of endogenous TNF-alpha little is known about the location and distribution of TNF-alpha-expressing cells at disease sites. The aim of the present work is, therefore, to investigate TNF-alpha and its cellular distribution in RAU lesions compared with those in induced oral traumatic ulcers (TUs). Twelve biopsies of RAU lesions of oral mucosa were obtained from 12 patients with RAU. They were compared to a control group consisting of ten samples of induced TUs. All samples were analyzed for TNF-alpha expression by using monoclonal mouse anti-human TNF-alpha antibody in avidin-biotin-peroxidase complex (ABC) staining. Results were quantified by a semi-automatic VIDAS image analysis system. TNF-alpha immunoreactivity was contained mainly in monocyte/macrophages and lymphocytes within the mononuclear inflammatory infiltrates. TNF-alpha was often seen in mast cells and vascular endothelial cells in connective tissue lateral to the inflammatory infiltrates. Interestingly, 32%-60% of the mononuclear cells were found to be TNF-alpha immunoreactive in RAU lesions. TNF-alpha containing cells were more numerous in aphthae (188+/-46 cells/0.2 mm2) compared with controls (52+/-14 cells/0.2 mm2, P<0.001). These findings suggest that RAU lesions are characterized by high expression of TNF-alpha. Because such expression occurred in the mononuclear inflammatory cells, mast cells and vascular endothelial cells, TNF-alpha, which is a major inflammatory mediator, may contribute to the activation and recruitment of leukocytes that are found in RAU lesions.

Elevated levels of interferon gamma, tumor necrosis factor alpha, interleukins 2, 4, and 5, but not interleukin 10, are present in recurrent aphthous stomatitis.

OBJECTIVE: To investigate our hypothesis that recurrent aphthous stomatitis (RAS), an inflammatory disease of the oral mucosa, is the result of an abnormal oral mucosal cytokine cascade leading to an enhanced cell-mediated immune response directed toward focal areas of the oral mucosa. DESIGN: Prospective nonrandomized case-control study. SETTING: Academic referral center PATIENTS: For part 1, 21 patients with RAS and 7 control patients; for part 2, 6 patients with RAS and 6 control patients. INTERVENTION: For study part 1, lesional and clinically normal oral mucosal biopsy specimens were obtained during an acute episode (within 72 hours of onset of ulcer) from 21 patients with RAS. Normal oral mucosal biopsy specimens were obtained from 7 healthy individuals, who served as controls. In study part 2, oral mucosal biopsy specimens were obtained from 6 RAS and 6 control patients at 24 and 48 hours after surgical trauma to those sites. MAIN OUTCOME MEASURES: Detection of the following messenger RNA (mRNA) types by use of semiquantitative reverse transcriptase polymerase chain reaction. For part 1, interleukins (IL) 2, 4, 5, and 10, interferon gamma, and tumor necrosis factor alpha were measured. For study part 2, IL-10 and interferon gamma were measured. RESULTS: In part 1, elevated levels of IL-2, interferon gamma, and tumor necrosis factor alpha mRNAs were detected in RAS lesions, consistent with a cell-mediated immune response. The IL-10 mRNA was not increased in RAS lesions. In addition, lower resting levels of IL-10 mRNA were detected in the clinically normal mucosa from patients with RAS, as compared with levels seen in the healthy controls. In part 2, at both 24 and 48 hours following trauma to the oral mucosa, the levels of mucosal IL-10 mRNA remained lower in patients with RAS than those observed in healthy controls, while interferon gamma mRNA levels were higher. CONCLUSION: Failure to suppress the inflammatory reaction initiated by trauma or other external stimuli, likely involving a functional deficiency of IL- 10 in the oral mucosa, appears to be important in the pathogenesis of RAS.

Raised anti-endothelial cell autoantibodies (AECA), but not anti-neutrophil cytoplasmic autoantibodies (ANCA), in recurrent oral ulceration: modulation of AECA binding by tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma).

Recurrent oral ulceration (ROU) is a common oral mucosal condition of unknown etiology. However, there is evidence to suggest that vasculitis may play a role. Here we investigate the presence in ROU of two autoantibodies associated with vasculitis, AECA and ANCA. AECA target as yet unidentified antigens on the endothelial cell surface and have been identified in patients with vasculitic disorders and inflammatory conditions with a vasculitic component. ANCA target specific neutrophil-associated proteins and are detected in specific vasculitic and chronic inflammatory disorders. AECA and ANCA levels were studied in 20 ROU patients and 20 controls. IgG AECA to the endothelial cell line ECV 304 were detected in 19 ROU patients and four controls. Levels were significantly raised in ROU both to ECV 304 (P < 0.000 05) and to human umbilical vein endothelial cells (HUVEC) (P < 0.005). Although levels were highest during episodes of ulceration, they were also raised between episodes. Stimulation of endothelial cells with TNF-alpha significantly increased AECA binding of both ROU (P < 0.005) and control samples (P < 0.0001), while IFN-gamma decreased binding (ROU P < 0.0001; controls P < 0.05). In contrast, ANCA were detected in only one patient and none of the controls. The presence of raised levels of AECA lends support to the hypothesis that a vasculitic process may underlie ROU. Moreover, these findings suggest that endothelial cell expression of AECA target antigens is increased by TNF-alpha and decreased by IFN-gamma stimulation.