Immunopathogenesis of oral submucous fibrosis by chewing the areca nut.

Oral submucous fibrosis (OSF) is a chronic, progressive, scarring, and premalignant disease of the oral mucosa. Its pathogenic factors are complex and include chewing areca nuts or other spicy food items, nutrition, and genetic and immune factors. Recently, immune factors have become the focus of medical research, with increased attention being paid to the role of immune regulation in diseases, particularly tumors. OSF is accompanied by obvious changes in the immune microenvironment. The aim of this review is to discuss the potential relationship of OSF and areca nuts genetic with the immune system, including lymphocytes, macrophage, Langerhans cell, mast cell, and substances released by activated immune cells, to determine the pathogenesis and treatment of OSF from an immunologic viewpoint.

Histo-Blood Group Antigens in Oral Cancer and Potentially Malignant Disorders.

BACKGROUND: Early detection of oral cancer is of critical importance because survival rates markedly improve when oral lesions are identified at an early stage. Aim of the present study is to investigate the expression of ABO (H) antigens in tissue specimens of oral cancer and potentially malignant disorders and to determine the role of ABO (H) antigens in tumour staging. MATERIALS AND METHODS: A prospective study was conducted on 60 cases of oral cancer and potentially malignant diseases. Specific red cell adherence test (SRCA-test) was used for studying A, B and O (H) antigens in tissue specimens and iso-antigenicity of epithelium was graded according to degree of adherence of indicator red blood cells. RESULTS: Among OSMF group, grade II adherence was seen in 53.3% cases, grade III in 33.3% cases, grade IV in 13.3% cases. In leukoplakia group, grade II adherence was seen in 26.7% cases, grade III adherence in 53.3% cases, grade IV adherence in 20% cases. Within the leukoplakia group, cases with dysplasia showed decreased adherence, compared with cases without dysplasia. Oral cancer group, negative adherence was seen in 13.3% cases, grade I adherence in 46.7% cases, grade II in 40% cases. In oral cancer group, antigen reactivity was less in poorly and moderately differentiated carcinoma, compared to well differentiated carcinoma. CONCLUSIONS: Antigen adherence and degree of loss of ABO (H) antigens in tissue specimens can be used for staging of the tumour.

Cys-X-Cys ligand 9 might be an immunological factor in the pathogenesis of oral submucous fibrosis and its concomitant oral lichenoid lesion.

OBJECTIVES: Oral submucous fibrosis (OSF) is a chronic oral precancerous disease primarily caused by betel quid chewing. Some OSF patients are concomitant with oral lichenoid lesion (OLL), a white-streak lesion with a higher risk for cancerization, in OSF mucosa. Immunological reaction has been considered as one of their common pathogenic mechanisms. Cys-X-Cys ligand 9 (CXCL9) is an important factor to recruit effector neutrophils and lymphocytes in immunological reactions. However, the expression levels of CXCL9 in OSF and OLL remain unclear. MATERIALS AND METHODS: We investigated the expression levels of CXCL9 in 10 normal buccal mucosa (NBM) samples and 56 OSF concomitant with OLL patients, and evaluated the possible mechanism of CXCL9 on their pathogenesis. RESULTS: Our results showed NBM demonstrated negative CXCL9 expression. OSF stained positive CXCL9 mainly in the cytoplasm of inflammatory cells and endothelial cells throughout the superficial layer of connective tissue, while its concomitant OLL showed much stronger CXCL9 in all mononuclear cells of subepithelial inflammatory infiltration (p = 0.0006). There was an upregulated trend of CXCL9 expression from NBM to OSF to OLL. However, no significant association between CXCL9 expression and clinicopathologic parameters of patients was found. CONCLUSIONS: In conclusion, CXCL9 was found for the first time to contribute to the immunological pathogenesis for both OSF and its concomitant OLL, indicating a continuously enhanced intensity of immunoreactivity in their pathogenic process. CLINICAL RELEVANCE: CXCL9 might be a useful tool to monitor the phase and disease severity of OSF and OLL, and a potential target for further clinical therapy for both lesions.

Salivary level of interleukin-8 in oral precancer and oral squamous cell carcinoma.

BACKGROUND: Interleukin 8 (IL-8) is a pro-angiogenic, pro-inflammatory mediator that belongs to the family of chemokines. Due to its pro-angiogenic characteristic, it may play a vital role in tumour angiogenesis and progression. OBJECTIVES: This study was designed to estimate the levels of salivary IL-8 in oral precancer and oral squamous cell carcinoma (OSCC) patients and compare them with healthy controls. The aim was to evaluate its efficacy as a potential biomarker for these diseases. MATERIALS AND METHODS: Each group comprised 25 individuals. The salivary IL-8 levels were determined by enzyme-linked immunosorbent assay. RESULTS: The levels of salivary IL-8 were found to be significantly elevated in patients with OSCC as compared to the precancer group (p < 0.0001) and healthy controls (p < 0.0001). However, the difference in salivary IL-8 concentrations among the precancer group and controls was statistically non-significant (p = 0.738). CONCLUSIONS: Our results suggested that salivary IL-8 can be utilised as a potential biomarker for OSCC. Salivary IL-8 was found to be non-conclusive for oral premalignancy in this preliminary study. Hence, its possible role in transition from premalignancy to malignancy needs further research with larger sample sizes. CLINICAL RELEVANCE: Saliva as a diagnostic biofluid offers a number of advantages over blood-based testing. The role of IL-8 in oral cancer if validated further by future research can provide an easy diagnostic test as well as a prognostic indicator for patients undergoing treatment. Therefore, if it's role in tumourigenesis can be sufficiently assessed, it could open up new avenues to find out novel treatment modalities for oral cancer.

Pathogenesis of oral submucous fibrosis.

Data from recent epidemiological studies provide overwhelming evidence that areca nut is the main etiological factor for oral submucous fibrosis (OSF). It is logical to hypothesize that the increased collagen synthesis or reduced collagen degradation is the possible mechanism in the development of the disease. There are numerous biological pathways involved in the above processes and it is likely that the normal regulatory mechanisms are either down regulated or up regulated at different stages of the disease. The copper content of areca nut is high and the possible role of copper as a mediator of fibrosis is supported by the demonstration of the up regulation of lysyl oxidase in OSMF biopsies. The aim of this article is to emphasize that the incorporation of copper into the areca nut is through the Bordeaux mixture, which is sprayed as a fungicide on areca plantations in regions with scheduled monsoons and of which copper sulfate is an important constituent.

Correlation of salivary and serum IgG, IgA levels with total protein in oral submucous fibrosis.

Oral submucous fibrosis (OSMF) is a disabling, potentially malignant condition of the oral cavity. The aetiology of OSMF is multifactorial but remains obscure. Although arecanut is considered to be the most important causative agent, responses observed in individuals using arecanut vary in relation to quantity and duration. It is considered that an immunological process is responsible for the pathogenesis of disease. We correlated salivary immunoglobulin A (IgA), salivary immunoglobulin G (IgG) and serum immunoglobulin A (IgA), serum immunoglobulin G (IgG), levels by turbidometric immunoassay. We estimated the levels of total serum protein (TSP) and haemoglobin (Hb) to determine the role of nutritional deficiency. The study population comprised 30 cases of OSMF and 10 controls. Five milliliters of blood and 2 ml of saliva were collected. Quantitative analysis of serum and salivary IgG, IgA was done by turbidometric immunoassay. TSP and Hb were estimated by Biuret and cyanmethaemoglobin methods, respectively. All patients showed significant (P < 0.01) increase in serum and salivary IgG, IgA levels as compared to controls. TSP patients showed significant (P < 0.01) decrease as compared to controls. Results of Hb in patients were not significant. The estimation of immunoglobulin levels is important to support the concept of autoimmune basis. Estimation of TSP and Hb suggests that nutrition has a definite role in OSMF.

Transforming growth factor-ß-1 polymorphisms are infrequent but exist at selected loci in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) may be considered a collagen metabolic disorder resulting from areca-nut alkaloid exposure and individual variation in collagen metabolism. Due to the complexity of OSF pathogenesis, it is important to elucidate independent and interactive effects of polymorphisms of collagen-related genes on OSF risk. MATERIALS AND METHODS: This study is focused on seven polymorphisms (SNPs) of transforming growth factor-beta-1 (TGF-beta-1) gene in patients with oral submucous fibrosis (OSF), belonging to south Indian ethnic extraction. The mean age at presentation was 43.9 years, range 23-72 years (n=50, M:F ratio, 2.6:1). DNA samples from 50 subjects of the same ethnic group and comparable demographic features who have had practiced the habit of areca-chewing of almost equal duration, but remained free of disease constituted the controls. All DNA samples were collected progressively and purified from peripheral blood employing standard protocols and tested for SNPs. They included two polymorphisms in the promoter region (C-509T and G-800A), three polymorphisms in exon-1 (Arg25Pro(G915C), Leu10Pro(T869C), Glu47Gly(A979G) and two in 5 ¢UTR regions (C→T(rs13306708) and G→A (rs9282871). The extracted DNA samples along with the primers underwent PCR amplification and the genotypic and allelic frequencies were calculated. All calculations were performed using the SPSS software. The PCR products were purified and subsequently sequenced using Flour S™ multi-imager system (Biorad). The sequenced data were analyzed using the BioEdit sequence analysis software. RESULTS: Out of the seven polymorphisms analyzed, six such as two in the promoter region, three in exon-1 and one in 5¢UTR were found to have a " P" value above 0.05 and hence were not significant. The C→T transition (rs13306708) in the 5¢UTR region recorded a " P" value of 0.03 on comparison and hence was found to be significant. The allelic frequencies for this C→T transition in patients were 68.7% C and 31.2% T (27CC, 15CT, 8TT) and that in controls were 89.5% C and 10.4% T (42CC, 6CT, 2TT). CONCLUSIONS: The polymorphism in 5¢UTR C-T in TGF beta 1 gene has a significant association with OSF, being a prime determinant in the pro-angiogenic pathway which has got direct bearing with the pathophysiology of the disease. The proximity of this polymorphism to the transcription site and the associated risk involved is discussed.

Cell-mediated immunity and head and neck cancer: with special emphasis on betel quid chewing habit.

Betel quid (BQ) chewing is popular in Taiwan, India, and many southeast-Asian countries. BQ chewing has strong association with the risk of oral leukoplakia (OL), oral submucous fibrosis (OSF), and oral cancer (OC). BQ components exhibit genotoxicity and may alter the structure of DNA, proteins and lipids, resulting in production of antigenicity. BQ ingredients are also shown to induce keratinocyte inflammation by stimulating the production of prostaglandins, TNF-alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes. These events may provoke tissue inflammation, early cell-mediated immunity (CMI), and immune surveillance in BQ chewers. However, BQ components also directly affect the functional activities of immunocompotent cells, and moreover tumor cells may hypo-respond to the CMI via diverse mechanisms such as induction of apoptosis of lymphocytes, induction of production of suppressor T cells, downregulation of MHC molecules in tumor cells, etc. Clinically, an alteration in lymphocyte subsets, a decrease in total number of lymphocytes, and a reduction in functional activities of CMI have been observed in isolated peripheral blood mononuclear cells (PBMC) and tumor infiltrated lymphocytes (TIL) in patients with OSF, OL or OC. Adaptation of tumor cells to immune system may promote clonal selection of resistant tumor cells, leading to immune tolerance. Future studies on effects of BQ components on CMI and humoral immunity in vitro and in vivo can be helpful for chemoprevention of BQ-related oral mucosal diseases. To elucidate how virus infection, tobacco, alcohol and BQ consumption, and other environmental exposure affect the immune status of patients with oral premalignant lesions or OC will help us to understand the immunopathogenesis of OC and to develop immunotherapeutic strategies for OC.

HLA typing in Taiwanese patients with oral submucous fibrosis.

BACKGROUND: A significant association of certain human leukocyte antigens (HLA) and haplotypic pairs with oral submucous fibrosis (OSF) has been reported. However, controversial result of no HLA association with OSF has also been reported. In this study, the phenotype and haplotype frequencies of HLA-A, -B, -C, -DRB1, and -DQB1 in 135 Taiwanese OSF patients were calculated and compared with those in 540 healthy control Taiwanese. METHODS: The analysis of HLA-A, -B, and -C antigens, and of HLA-DRB1 and -DQB1 alleles in OSF patients and healthy control subjects, was performed by serologic typing and DNA typing using polymerase chain reaction with sequence-specific primers (PCR-SSP), respectively. RESULTS: We found that the phenotype frequency of HLA-B76 (3.0%) in OSF patients was significantly greater than that (0%) in healthy control subjects (corrected P (Pc) = 0.000). In addition, the haplotype frequencies of HLA-B48/Cw7 (3.0%), -B51/Cw7 (6.7%), and -B62/Cw7 (8.2%) in OSF patients were significantly greater than the corresponding haplotype frequencies (0, 0.7, and 1.9%, respectively) in healthy control subjects (Pc = 0.000). The relative risk (RR) values of haplotypes B51/Cw7 (9.57) and B62/Cw7 (4.7) were greater than the RR values of phenotypes B51 (1.81), B62 (2.31), and Cw7 (1.91) in OSF patients. In addition, the etiologic fraction (EF) value of haplotype B51/Cw7 (0.63) was higher than the EF values of phenotypes B51 (0.2) and Cw7 (0.59). CONCLUSIONS: We conclude that some Taiwanese areca quid (AQ) chewers with particular HLA phenotypes and haplotypes are prone to have OSF. In addition, some particular HLA haplotypes may play more important roles than the individual HLA phenotypes for the genetic susceptibility to OSF. However, the significantly increased HLA phenotype B76 and three of the common HLA haplotypes detected are present in only about 20% of incident cases of OSF.

Levamisole and Chinese medicinal herbs can modulate the serum interleukin-6 level in patients with recurrent aphthous ulcerations.

BACKGROUND: Recurrent aphthous ulcerations (RAU) are common oral inflammatory lesions. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that has effects on cellular and humoral immunities. Previous studies have shown that the high serum IL-6 levels in some RAU patients can be reduced by drug treatment. This finding suggests that IL-6 may be a useful marker in evaluating therapeutic effects of RAU. METHODS: In this study, we used a solid phase, two-site sequential chemiluminescent immunometric assay to determine the baseline serum levels of IL-6 in a group of 228 patients with RAU, erythema multiforme (EM), traumatic ulcers (TU), oral submucous fibrosis (OSF), pemphigus vulgaris (PV), or Sjögren's syndrome (SS), and in 77 normal control subjects. Some RAU patients were treated with levamisole plus Chinese medicinal herbs or levamisole only for 0.5-5 months and their serum IL-6 levels were measured after treatment. RESULTS: We found that about 99% of the normal control subjects and the patients with EM, TU, or OSF had a serum IL-6 level within the normal limit of 5.0 pg/ml. However, 24% (48/197) RAU patients, 14% (1/7) EM patients, 43% (3/7) PV patients, and 100% (6/6) SS patients had a serum level of IL-6 greater than 5.0 pg/ml. The mean serum level of IL-6 in patients with RAU (3.6 +/- 3.5 pg/ml, P < 0.001), minor type RAU (2.7 +/- 2.0 pg/ml, P < 0.05), major type RAU (5.2 +/- 4.6 pg/ml, P < 0.001), or herpetiform type RAU (4.1 +/- 3.8 pg/ml, P < 0.01) was higher than that in normal control subjects. The mean serum level of IL-6 in major type (P < 0.001) or in herpetiform type RAU patients (P < 0.05) was higher than that in minor type RAU patients. The mean reduction of serum IL-6 level (10.0 +/- 7.1 pg/ml) in RAU patients after treatment with levamisole plus Chinese medicinal herbs was significantly higher than that (5.1 +/- 3.7 pg/ml) in RAU patients after treatment with levamisole only (P < 0.005), suggesting that the combination therapy is superior to the single therapy of levamisole only. CONCLUSION: We conclude that levamisole and levamisole plus Chinese medicinal herbs can modulate the serum IL-6 level in RAU patients. Although the therapeutic effect of RAU can be assessed by a decrease in the frequency, duration and number of the oral ulcerations, it can also be monitored by a reduction of serum IL-6 level in RAU patients.

High incidence of autoantibodies in Taiwanese patients with oral submucous fibrosis.

BACKGROUND: Previous study has shown a high incidence of autoantibodies including antinuclear (ANA), antismooth muscle (SMA), antigastric parietal cell (GPCA), antithyroid microsomal (TMA), and antireticulin antibodies in a small group of 26 patients with oral submucous fibrosis (OSF). The reasons why some of the OSF patients have high titers of autoantibodies in serum have not been completely explained and no further study on autoantibodies in OSF patients has been done in a large group of patients. METHODS: In this study, we determined the serum levels of ANA, SMA, GPCA, and TMA in a large group of 109 male Taiwanese patients with OSF by an indirect immunofluorescence technique (for ANA, SMA, and GPCA), and by a semiquantitative microtiter particle agglutination test (for TMA). The presence of serum autoantibodies in OSF patients was further correlated with patients' oral habits and the severity of OSF measured by maximum mouth opening (MMO) and sites of involvement. RESULTS: We found that the frequencies of presence of serum ANA (23.9%), SMA (23.9%), and GPCA (14.7%) in OSF patients were significantly higher than those (9.2, 7.3, and 5.5%, respectively) in healthy control subjects (P < 0.01, P < 0.005, and P < 0.05, respectively). Although the frequency of presence of TMA (5.5%) in OSF patients was also greater than that (2.8%) in healthy control subjects, the difference was not significant (P > 0.05). The presence of serum GPCA in OSF patients was significantly associated with daily areca quid (AQ) consumption (P < 0.05). The presence of serum ANA in OSF patients associated with daily AQ consumption was of borderline statistical significance (P = 0.066). However, no significant correlations were demonstrated between the presence of serum autoantibodies in OSF patients and other variables of oral habits, MMO, and sites of involvement. CONCLUSION: In this study, all the 109 OSF patients had AQ chewing habit and 73.4% of the OSF patients swallowed the 'juice' of AQ during the chewing process. The presence of serum GPCA and ANA in OSF patients was associated with daily consumption of AQs. AQ chewing caused mucosal microtrauma, and ulcerations facilitated the diffusion of genotoxic and cytotoxic AQ ingredients into the oral and gastric tissues. Altered autoantigens released from AQ ingredients-damaged cells may induce autoantibody production. Higher frequencies of specific HLA-DR antigens in OSF patients may also help autoantibody production. Therefore, we conclude that the high incidence of autoantibodies in OSF patients may be due to AQ chewing habit, toxic AQ ingredients, and genetic susceptibility of the OSF patients.

Quantitative analysis of immunocompetent cells in oral submucous fibrosis in Taiwan.

Previous studies have shown that the local and systemic upregulation of inflammatory and fibrogenic cytokines and downregulation of antifibrotic cytokines are central to the pathogenesis of oral submucous fibrosis (OSF). The immunocompetent cells, especially the macrophages and lymphocytes, are likely the main source of cytokine synthesis. Therefore, this study used an immunohistochemical method to quantify the T lymphocyte, B lymphocyte and macrophage densities in the epithelium and subepithelial connective tissue of 50 specimens of moderately advanced and advanced OSF and 10 specimens of normal oral mucosa (NOM). The mean T lymphocyte, B lymphocyte and macrophage densities in OSF specimens were 555.2+/-417.4, 63.4+/-44.3 and 66.9+/-76.4 cells/mm(2) in the subepithelial connective tissue and 308.1+/-261.1, 1.4+/-3.5 and 6.6+/-11.9 cells/mm(2) in the epithelium, respectively. These findings suggest that T lymphocytes were the major immunocompetent cells in both the subepithelial connective tissue and epithelium of OSF specimens. Macrophages and B lymphocytes are the minor immunocompetent cells in the subepithelial connective tissue and are only occasionally found in the epithelium of OSF specimens. Similar distribution of immunocompetent cells was also found in NOM specimens. However, the mean T lymphocyte, B lymphocyte and macrophage densities in the subepithelial connective tissue (271.2+/-107.0, 13.3+/-18.4 and 17.3+/-19.1 cells/mm(2), respectively) and the mean T lymphocyte density in the epithelium (97.7+/-51.4) of NOM specimens were significantly lower than the corresponding mean cell densities in OSF specimens. Using frozen tissue sections, we further quantified the CD4+ and CD8+ lymphocyte numbers in eight moderately advanced or advanced OSF specimens. It was found that the CD4+ and CD8+ lymphocyte densities were 213.3+/-140.7 and 101.5+/-72.8 cells/mm(2) in the subepithelial connective tissue of OSF specimens, respectively. The CD4+ to CD8+ lymphocyte ratio was 2.1:1. Our results showed a significant increase in the number of T lymphocytes and macrophages and a predominance of CD4+ lymphocytes over CD8+ lymphocytes in the subepithelial connective tissue of OSF specimens. We conclude that the cellular immune response may play an important role in the pathogenesis of OSF.

Visceral organ involvement is infrequent in oral submucous fibrosis (OSF).

Associated visceral organ involvement evidence by systemic fibrosis has not been explored in oral submucous fibrosis (OSF). The investigations in this aspect were limited to loco-regional sites of naso/oropharynx and oesophagus. The study of whether the oral fibrosis is part of a systemic spectrum of disease involving multiple organs is an interesting pursuit. With this intention the patients diagnosed on clinical and histological grounds for OSF were concurrently tested by biophysical means for the presence of endomyocardial fibrosis (EMF), pancreatic (PF) and retroperitoneal fibrosis (RPF), which are endemic to the area studied. Twenty-five (n = 25) cases of OSF who visited the Department of Oral pathology & Microbiology. Govt. Dental College, Trivandrum, India for symptomatic relief of their illness comprised the study group. Ten (n = 10) age and sex matched healthy volunteers comprised the control. All the subjects have had undergone cardiologic and gastrointestinal investigations to rule out the possibility of concurrent EMF and PF. The patients were all of Indian ethnic extraction and mostly (> 90%) were from low socio economic classes. The mean age of the patients was 54.16 +/- 14.6 years, including 18 females and 7 males (F:M = 2.57:1). The severity of fibrosis was unrelated to the age of patients (P > 0.05). All the patients were chewers of areca quid (12%)/tobacco (88%). In addition to quid chewing 3/25 (12%) patients smoked 'bidi' and 6/25 (24%) consumed home brewed liquor (arrack/toddy) which contain about 40-50% ethanol. Statistically no relationship was observed between the clinical stages of OSF and severity of epithelial dysplasia in this study (P > 0.05). Out of the 25 patients, 5 (20%) showed sclerotic aortic value which may be an age related finding. Also 7 (28%) patients were found to be hypertensive and interstitial lung disease was present in 2 (8%). The possibility of EMF in one female patient who showed thickened RV apical endocardium was ruled out by cardiac catheterisation. Thus none of the patients showed evidence of endomyocardial fibrosis. The pancreas was found to be hyperchoic in 8(32 1/4) by ultra sonography. Liver was found to be hyperchoic in 6 (24%). Fat stain in stool samples was found to be positive in 13(58%). The hyperchogenecity of pancreas may be due to alcoholism or an underlying endocrine pancreatic insufficiency like diabetes and not due to pancreatic fibrosis. The positivity of fat stain could be due to fatty liver/alcoholism. Thus the study fails to reveal any evidence of pancreatic fibrosis in the group. The lack of any evidence of an associated visceral organ fibrosis in OSF made it prudent to believe that this is a loco-regional disease, initiated by local factors and propagated under their influence without systemic involvement.

Oral submucous fibrosis patients have altered levels of cytokine production.

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in betel quid chewers and is characterised by dense bands of collagen in the juxta-epithelial region preceded by inflammation. We have investigated the spontaneous and stimulated production of cytokines by peripheral blood mononuclear cells (PBMC) from OSF patients and compared them with genetically-related relatives, Indian and Caucasian control subjects. The cytokines studied included: interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The results show: a) significant differences in the stimulated versus non-stimulated levels of IL-1beta, IL-6, IL-8 and TNF-alpha but not of IFN-gamma production by patients, and in the relatives' stimulated versus non-stimulated levels of IL-1beta, IL-6 and IFN-gamma; b) no difference in the spontaneous cytokine production between any two groups; and c) significant increases in the patients' stimulated cytokines compared to the Caucasian and Indian controls (P< or =0.050). These results demonstrate increased levels of proinflammatory cytokines and reduced anti-fibrotic IFN-gamma in patients with OSF, which may be central to the pathogenesis of OSF.

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis.

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although oversimplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.

An immunohistochemical study of oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic disease of the oral cavity characterized by inflammation and progressive mucosal fibrosis. These reactions may be the result of either direct stimulation from exogenous antigens like areca alkaloids or by changes in tissue antigenicity that may lead to an autoimmune response. This study investigated the presence and distribution of inflammatory cells and MHC class II antigen expression by epithelial and immunocompetent cells using a three-stage immunoperoxidase method on frozen sections. Thirty OSF tissue specimens and ten normal buccal mucosae were studied and compared. All tissues were investigated using antibodies to T cells (CD3), T helper/inducer cells (CD4), T suppressor/ cytotoxic cells (CD8), B cells (CD20), naive T cells and monocytes (CD45RA), macrophages, Langerhans' cells (CD68) and HLA-DR-positive cells (HLA-DR alpha). The predominant cell populations detected in normal tissues were CD3, CD4 and HLA-DR-positive cells. The distribution of CD4-positive cells was similar to that of CD3-positive cells, which were scattered, often uniformly distributed, both in the epithelium and connective tissue. CD8-positive cells were occasionally seen in the normal epithelium and lamina propria. Few scattered B cells (CD20) and macrophages (CD68) were observed in normal mucosa. Naive T cells (CD45RA) were seen in all normal tissues focally concentrated around the connective tissue papillae, with a similar distribution to that of CD3-positive cells. All normal sections showed HLA-DR-positive cells scattered both in the epithelium and in the lamina propria. Epithelial cells did not show any positive reaction to this antibody and many intraepithelial positive cells showed a dendritic morphology. The cell population detected in OSF showed higher numbers of CD3 and HLA-DR-positive cells compared with those of the normal tissues. The pattern of staining for CD4-positive cells in OSF tissues was similar to that of CD3-positive cells both in the epithelium and connective tissue and was higher than that in normal tissues. A few scattered CD8-positive cells and only occasional CD20- and CD68-positive cells were seen in OSF sections. Few CD45RA-positive cells were found in the epithelium and lamina propria of OSF sections. However, OSF specimens showed high numbers of HLA-DR-positive cells in the basal layer of the epithelium, juxtaepithelium and in the lamina propria in a similar distribution to that of CD3 cells compared with the normal tissues. Most HLA-DR-positive cells in the epithelium showed dendrites directed vertically towards the surface. The increased evidence of CD4 and HLA-DR-positive cells in OSF tissues suggests that most lymphocytes were activated and shows an increased presence of Langerhans' cells. The presence of these immunocompetent cells and high ratio of CD4 to CD8 in OSF tissues suggest an ongoing cellular immune response leading to a possible imbalance of immunoregulation and alteration in local tissue architecture.

Immunological studies in oral submucous fibrosis.

Oral submucous fibrosis (OSMF) is a high risk precancerous condition. The possible role of immunological factors in the pathogenesis of this condition was evaluated in 113 cases and 25 controls. The male/female ratio was 1.5/1. The mean age of males was significantly lower than that of females. The mean ESR levels were within normal limits, but for a higher than 20 mm fall per hr. in 40% of the cases. The serum IgA, IgG, and IgM levels were elevated significantly as compared to the controls. Circulating auto-antibodies and tissue-deposited antibodies were also found in 33% and 40% of the cases, respectively. From the analysis of the results, it is difficult to ascribe an auto-immune basis for the causation of OSMF. The female bias and elder age group, the factors generally considered in favour of an immune disorder, was not found in our study. However, raised ESR in 40% and serum globulin levels in 47% of the patients, distinctly higher levels of serum immunoglobulins, and positivity for circulating and tissue deposited antibodies in 33% and 34% of the cases respectively, do indicate an immunological basis. Therefore, further studies are required to ascertain the role of cellular immune mechanism and genetic parameters to explain the etiopathogenesis of this complex clinical entity.

HLA-antigens in oral submucous fibrosis.

HLA-typing was carried out on 122 areca nut chewers who attended hospitals for complaints unrelated to the habit. The subjects were South Africans of Indian extraction. The study did not include haplotypes. Palpable fibrous bands in the mouth indicated oral submucous fibrosis. The subjects were divided into 4 groups based on specific oral symptoms and signs. Groups A and B were without fibrous bands. Group A (47 subjects) included those with one or no symptoms while group B (28 subjects) suffered from 2 to 7 oral symptoms. Group C (17 subjects) had oral symptoms and represented early or mild oral submucous fibrosis and exhibited at least one discrete palpable fibrous band. Group D (30 subjects) were classic oral submucous fibrosis cases with multiple bands. The high occurrence of oral submucous fibrosis in this study group (39%) is similar to the occurrence in comparable age groups reported earlier in South Africa and is conceivably due to the higher age range of the subjects and their relatively long exposure to the areca nut. We were unable to demonstrate a specific pattern of HLA-antigen frequencies in chewers with or without the disease. Furthermore, there were no differences between the study population and the controls. It is concluded that there is not necessarily a HLA-associated susceptibility in oral submucous fibrosis.

Pathogenesis of oral submucous fibrosis. Relationship to risk factors associated with oral cancer.

Oral submucous fibrosis (OSF) is a chronic disease of the oral mucosa characterized by inflammation and a progressive fibrosis of the lamina propria and deeper connective tissues. It is a condition predominantly seen among people of Indian origin, and an epidemiologic survey done a decade ago showed no less than 250,000 cases in India, a figure that must have increased sharply. OSF is a condition with a high risk of malignant transformation; to date, no conclusive etiologic agent has been identified, although plenty of data have been generated on various aspects of the disease. These include genetic, carcinogenic, immunologic, viral, nutritional, and autoimmune possibilities, all of which also have been implicated in the development of oral cancer. This article reviews existing evidence on the pathogenesis of OSF and its relation to oral cancer and suggests a possible multifactorial model to explain the disease process.