The etiology of oral leukoplakia has not been elucidated. A previous study using in situ staining with a DNA-binding fluorescent dye (Hoechst 33258) demonstrated numerous small granular structures in the cytoplasm of the epithelial cells in leukoplakia, suggesting that microorganisms were present. Furthermore, it was speculated that the microorganisms in the epithelial cells were oral mycoplasma. In this study, to examine intracellular infection by mycoplasma in epithelial cells with leukoplakia, fluorescent immunohistochemistry was performed using anti-mycoplasma rabbit polyclonal antibodies. Thirty-one formalin-fixed, paraffin-embedded specimens of leukoplakia without epithelial dysplasia and 19 epithelial dysplasia specimens were examined. Normal appearing mucosa specimens were used as controls. In all specimens of leukoplakia without epithelial dysplasia, numerous small granular fluorescing structures were observed within the cytoplasm of epithelial cells. In 10 of 19 (52.3%) specimens with epithelial dysplasia, fluorescing structures were observed, but were not observed in the remaining 9 specimens. In normal-appearing mucosa specimens, a few or no fluorescing structures were observed. These findings strongly suggest that mycoplasma infection is related to hyperkeratosis of oral mucosa. However, the relation between mycoplasma infection and epithelial dysplasia could not be clarified.
Oral leukoplakia is the most common premalignant lesion of the oral mucosa, although its etiology has not yet been elucidated. Hoechst 33258, DNA binding fluorescent dye, staining of epithelial cells from oral leukoplakia tissues has revealed small, granular, and fluorescent structures in the cytoplasm, which are suggestive of microorganism infection. The Hoechst 33258-stained particles have been speculated to be mycoplasmas. Some mycoplasmas, such as Mycoplasma salivarium and Mycoplasma orale, form part of normal flora of the oral cavity, and can be detected in dental plaque and saliva. So, this study was performed to examine the location of mycoplasma in the epithelial cells of oral leukoplakia tissue by immunohistochemistry, immunoelectron microscopy, nested polymerase chain reaction (PCR) and sequencing of nested PCR products. By immunohistochemistry, small, granular fluorescent structures in the cytoplasm of epithelial cells were observed. The results of immunoelectron microscopy demonstrated the presence of small (0.5-0.8μm in size) polymorphous, electron dense structures with definite borders. Their inner structures were relatively homogeneous and their borders were definite. And binding of gold particles was observed limitedly on the structures. The results of nested PCR and sequencing of PCR products showed the presence of M. salivarium in the epithelial cells of three leukoplakia tissues. From these findings, invasion of M. salivarium in the oral mucosal epithelial cells of leukoplakia is shown.