Yoshihiro Abiko

Defensins in saliva and the salivary glands

Saliva contains several types of antimicrobial peptides that play a role in innate immunity. Peptides that were recently added to this list are the defensins. The purpose of this review is to summarize what is known about the production and role of defensins in the salivary glands and to discuss their therapeutic potential. The Α-defensins, human neutrophil defensins (HNP)-1, -2, and -3, have been detected in saliva and may be derived from neutrophils. The Β-defensins, human Β-defensins (HBD)-1 and -2, have also been detected in saliva. Although it has been speculated that salivary HBDs are derived from keratinocytes that line the oral mucosa rather than from the salivary glands, the HBD-1 peptide was recently found to be specifically expressed in salivary ductal cells, although not in acini. Defensins may be useful for the treatment of periodontal disease and for the prevention of caries and periodontitis.

Cell Death of Osteocytes Occurs in Rat Alveolar Bone during Experimental Tooth Movement

The purpose of this study was to examine the morphological changes in alveolar bone osteocytes on the pressure side during experimental tooth movement, using quantitative evaluation on hematoxylin and eosin-stained sections, the TUNEL method, confocal laser scanning microscopy (CLSM), and transmission electron microscopy. In 8-week-old Wistar rats, the left first molar was forced to move mesially with an average load of 10 g by a nickel-titanium superelastic wire. After 6 hours, nuclear condensation and fragmentation appeared in osteocytes adjacent to the hyalinized periodontal ligament (PDL). These cells showed TUNEL-positive reaction. The number of osteocytes with apoptosis progressively increased up to 1 day. At 1 and 2 days, cytoplasmic and nuclear destruction and distribution within the lacunae occurred and increased up to 4 days. The proportion of necrotic osteocytes and near empty lacunae peaked at 2 and 4 days, respectively. At 7 days, necrotic osteocyte and empty lacunae numbers returned to the level of control bone, probably due to resorption of the alveolar bone containing apoptotic and necrotic osteocytes. Ultrastructually, the osteocytes showed apoptotic morphology at 6 and 12 hours and 1 day; at 2 and 4 days, several osteocytes exhibited characteristics of necrosis and destructive images of the surrounding bone matrix, which resulted in enlargement of the lacunae. The present results demonstrate that osteocytes in alveolar bone adjacent to the hyalinized PDL underwent cell death via apoptosis and secondary necrosis during orthodontic tooth movement, which may be associated with the subsequent bone resorption.

Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection

Defensins are small cationic proteins that harbor broad-spectrum microbicidal activity against bacteria, fungi and viruses. This study examines the effects on pathogens of the epidermis engineered to express human beta-defensin 3 (HBD3) to combat bacterial infections. First, we examined the localization of HBD3 in the epidermis and observed HBD3 in the intercellular spaces and lamellar bodies of the upper epidermal layers. This result showed HBD3 expressed and assembled in the outer layers of the epidermis was suspected to counter the invading microorganisms. Next, we established a keratinocyte cell line that stably expressed HBD3 and found that the culture medium showed antibacterial activity. Furthermore, we prepared an epidermal sheet of these cells with the HBD3 gene and grafted this onto a dermal wound on a nude rat. The HBD3 engineered epidermis demonstrated significant antimicrobial activity. Skin ulcers without epidermis are constantly exposed to invading microorganisms. Biopsy samples of re-epithelizing epidermis from patients with skin ulcers were collected, and HBD3 mRNA level measured in the epidermis. The epidermal samples from the ulcer skin expressed 2.5 times higher levels of HBD3 transcript than those in the control skin. These results, taken together, indicate that the therapeutic introduction of the HBD3 gene into somatic cells may provide a new gene therapy strategy for intractable infectious diseases.

Expression of MIP-3α/CCL20, a macrophage inflammatory protein in oral squamous cell carcinoma

Abstract We have examined the expression of MIP-3α/CCL20 in oral squamous cell carcinoma (SCC) in vivo and in vitro. In addition, we have investigated whether the expression of MIP-3α/CCL20 is regulated by bacterial infection and inflammatory cytokines. In order to determine the mRNA level of MIP-3α, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with LightCycler™ using the double-stranded DNA dye, SYBR Green I. Oral epithelial cells and six SCC cell lines (SCC-9, SAS, BSC-OF, HSC-4, HSC, Ca9-22) were found to express MIP-3α mRNA. The expression of MIP-3α was upregulated by infection with Actinobacillus actinomycetemcomitans and by stimulation with lipopolysaccharide and TNF-α. By in situ hybridization, the detectable MIP-3α expression in SCC was localized primarily at the epithelial pearls corresponding to the spinous layer. These results suggest that MIP-3α contributes to the oral immunoresponse to bacterial infection, and may be involved in the growth of SCC.

Upregulation of Human Beta-Defensin 2 Peptide Expression in Oral Lichen Planus, Leukoplakia and Candidiasis. An Immunohistochemical Study

Human beta defensin 2 (hBD-2) is a major antimicrobial peptide that is produced by many types of epithelial cells, and is transcriptionally inducible by various proinflammatory agents, such as cytokines and bacteria. Although in vitro studies of the hBDs in oral epithelial cells have been well documented, only little is known about the in vivo pathological state of oral epithelium. We investigated the localization of hBD-2 peptide in tissue sections of oral lichen planus, leukoplakia, candidal leukoplakia and radicular cysts using immunohistochemistry. HBD-2 was stained in both the hyperkeratinized and the granular layers in cases of lichen planus with hyperkeratosis and leukoplakia. Expression in spinous and suprabasal layers was often strong in lichen planus. There were no significant differences in the number of S-100 positive dendritic cells between the widely stained areas and those with limited staining areas in lichen planus. In cases of candidal leukoplakia, the hyphae of candida were mainly detected on the surface of keratinization, which showed only negative or faint staining for hBD-2. These results suggest that hBD-2 is vigorously induced by lichen planus-related inflammation and that it plays an important role in protection from Candida albicans infection; however, it is not a strong chemotactic attractant for Langerhans cells in pathological conditions of oral epithelium.

Expression of inflammatory cytokines and beta-defensin 1 mRNAs in porcine epithelial rests of Malassez in vitro.

In the present study, we investigated the mRNA expression of inflammatory cytokines, including interleukin (IL)-1α, IL-6, IL-8, and granulocyte macrophage colony-stimulating factor (GM-CSF), and β defensin 1 (BD-1), an antimicrobial peptide, in the epithelial rests of Malassez in vitro. A reverse transcription-polymerase chain reaction (RT-PCR) assay was performed in order to observe the expression of these mRNAs. The effect of lipopolysaccharide (LPS) on the mRNA expression was also studied by quantitative RT-PCR assay, with a LightCycler, using the double-stranded DNA dye SYBR Green I. The mRNAs of the four kinds of inflammatory cytokines and BD-1 were detected in the epithelial cells under normal culture conditions. Immunocytochemical staining showed the expression of CD14, a receptor for LPS, on the epithelial cells. The mRNA expressions of IL-1α, IL-6, IL-8, and GM-CSF were upregulated by stimulation with LPS, in a dose- and time-dependent manner. Epithelial cells incubated with 1000 ng/ml of LPS for 6 h showed the most significant upregulation of the cytokine mRNAs. On the other hand, no obvious alteration of BD-1 expression by LPS stimulation was observed. The results indicated that the epithelial rests of Malassez may actively participate in the inflammatory response to bacterial infection, and that they play an important role in the defense mechanism of the radicular cyst.

Differential expression of human beta-defensin 2 in keratinized and non-keratinized oral epithelial lesions ; immunohistochemistry and in situ hybridization

Abstract. Human β-defensin(hBD)-2 , an antimicrobial peptide, is produced by various epithelial cells. Because hBD-2 expression in the oral epithelium has not been assessed, we investigated its localization in normal oral epithelium and epithelial lesions. hBD-2 expression was studied using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded tissue sections from 30 cases of squamous cell carcinoma and 6 cases of leukoplakia. Immunostaining for hBD-2 was more intense in hyperkeratinized than in ortho- or non-keratinized epithelium. In contrast, signals for hBD-2 mRNA were frequently stronger in non-keratinized epithelium than in hyper- or ortho-keratinized epithelium. The results suggest that keratinization in oral epithelium plays an important role in the biological function of hBD-2 both at the mRNA level and in the retention of the peptide in the epithelium.

Salivary Defensins and Their Importance in Oral Health and Disease

Saliva contributes significantly to the protective barrier of oral epithelium through its mechanical rinsing action and the unique peptides it contains. Saliva contains several types of antimicrobial peptides, including defensins, which may have an important role in innate host defense. Many types of human defensins have been discovered and characterized in the last decade. This review summarizes the recent literature on salivary defensins and discusses their importance in oral health and disease. Salivary defensins are possibly derived from salivary ductal cells, oral epithelial cells and some blood cells. The antimicrobial activity of defensins may be affected by the components of saliva. Salivary defensin levels can be altered in oral diseases, and therefore may be a useful marker for risk assessment, salivary diagnosis and therapeutic strategies.

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin.

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelilal nests.

Localization of human β-defensin 3 mRNA in normal oral epithelium, leukoplakia, and lichen planus: an in situ hybridization study

u2002Human β-defensin 3 (hBD-3), an antimicrobial peptide, is produced by various epithelial and some nonepithelial tissues. hBD-3 mRNA is widely expressed in oral tissues, including oral epithelium and the salivary glands. Although the localization of hBD-1 and hBD-2 has been well demonstrated in tissue sections, the localization pattern of hBD-3 has not yet been shown. In the present study, we investigated the expression pattern of hBD-3 mRNA by in situ hybridization using specific RNA probes; the signal for hBD-3 was detected in upper spinous and granular layers in normal oral epithelium. In cases of leukoplakia, a strong signal of hBD-3 mRNA was observed in the granular layer. In lichen planus, the signal was strongly detected in the spinous and suprabasal layers. The signals were stronger than those of either normal oral epithelium or leukoplakia. The results indicate that the localization pattern of hBD-3 is very similar to that of hBD-2. hBD-2 and hBD-3 may function together or compensate each other for expressional loss.