Kaoru Kusano

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.

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

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.

Upregulated expression of human β defensin-1 and -3 mRNA during differentiation of keratinocyte immortalized cell lines, HaCaT and PHK16-0b

BACKGROUND Human beta-defensins (hBDs) belong to a group of antimicrobial peptide that are expressed in the epithelial cells. OBJECTIVE The present study investigated mRNA expression levels of the beta-defensins, hBD-1, -2 and -3, in human keratinocytes during differentiation in vitro. METHODS Immortalized keratinocyte cell lines, HaCaT and PHK16-0b, were used in this study; in order to stimulate differentiation, the Ca(2+) concentration in the growth media was increased from 0.3 to 1.8 mM. RESULTS Four days after the increase, the expression levels of hBD-1 and -3 were increased in both cell lines, followed by an increase in the mRNA levels of the differentiation markers, involucrin and keratin 10. No increased expression of hBD-2 was observed. CONCLUSION The results indicate that keratinocyte differentiation may stimulate hBD-1 and -3 expression in stratified squamous epithelia.

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

 Human 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.

Single-nucleotide Polymorphism (SNP) in β-Defensin 2 in a Japanese Population and an Effect of - 1029 SNP on Promoter Activity

Abstract Human β-defensin (hBD) 2 is an epithelial antimicrobial peptide. We studied single-nucleotide polymorphisms in the gene of hBD-2 in a Japanese population, and estimated the effect of a polymorphism in the promoter/enhancer region on the transcriptional activity. By sequencing the hBD-2 gene of 50 unrelated individuals, we detected one SNP in exon 2 and nine SNPs in the promoter/enhancer region. The SNP in the coding region at the + 1765 position is synonymous [CCC (Pre)→CCT (Pre)]. One SNP in the promoter region (-1029) is located at the consensus sequence for NF-IL6 binding. By luciferase reporter assay and electrophoretic mobility shift assay, the wild-type (G) of -1029 showed significantly lower transcriptional activity than did the variant-type (A). The SNP at position -1029 may influence the hBD-2 expression and cause genetic variations in susceptibility to infectious diseases.