Indoleamine 2,3-dioxygenase and trophoblast invasion in caesarean scar pregnancy: Implications for the aetiopathogenesis of placenta accreta spectrum.

Immunohistochemical localisation of indoleamine 2,3-dioxygenase was studied in order to better understand the pathophysiology of placenta accreta spectrum. In the decidua staining for indoleamine 2,3-dioxygenase was found in the glandular epithelium with some additional positive cells. Extravillous cytotrophoblast invasion was present in the myometrium which was not covered by the decidual tissue whereas myometrial invasion of cytotrophoblasts was absent where this tissue lay deep to decidua. These results suggest that indoleamine 2,3-dioxygenase expression in the decidua may normally control trophoblast invasion and absence of its expression where decidua is absent may be involved in the pathogenesis of the over-invaded placenta.

Polymorphism of salivary histatin gene and periodontal disease in the Japanese population.

Chronic periodontitis is a widespread and major dental disease. Recent studies have analyzed a possible relationship between polymorphism of several genes and periodontitis. Histatins are salivary polypeptides with fungicidal activities against Candida albicans and yeast and bactericidal activities against Porphyromonas gingivalis and Streptococcus mutans. Histatins are part of the innate defense of the oral cavity. We examined the frequency of the polymorphism codon 23 of the histatin 3 gene (HIS2 allele) in relation to periodontitis in the Japanese population. The subjects were 143 Japanese individuals, of which 63 were healthy control subjects and 80 were periodontal patients. We isolated genomic DNA from lingual mucosal cells and tested them for single nucleotide polymorphism (SNP) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The incidence of polymorphism was analyzed statistically by Fisher's exact test. The results indicated that the gene polymorphism at codon 23 of the histatin 3 gene was not associated with periodontitis in the Japanese population (p = 0.166). Rather, if at all, it appeared to be associated with resistance to periodontitis.

Cooperation of salivary protein histatin 3 with heat shock cognate protein 70 relative to the G1/S transition in human gingival fibroblasts.

Histatins, a family of salivary proteins, have antimicrobial activity. Candida albicans, which is killed by histatins, induces oral candidiasis in individuals with compromised immune systems. Although the functional significance of histatins has been documented, their biological and physiological functions against host cells have not been clarified. In this study, we found that histatin 3, a member of the histatin family, binds to heat shock cognate protein 70 (HSC70). These proteins were co-localized in the cytoplasm and nucleus in human gingival fibroblasts following non-heat and heat shock. Histatin 3 induced stimulation of DNA synthesis and cell survival in human gingival fibroblasts in a dose-dependent manner. This DNA synthesis was found to be dependent on HSC70 by knockdown experiments. The effect of heat shock on DNA synthesis induced by histatin 3 was approximately 2-fold higher than that of non-heat shock. When the histatin 3 uptake into cells was inhibited by monodansylcadaverine or when histatin 3 binding to HSC70 was precluded by 15-deoxyspergualin, DNA synthesis by histatin 3 was approximately 2-fold less than that without monodansylcadaverine or 15-deoxyspergualin. Although HSC70 directly bound to p27(Kip1) (a cyclin-dependent kinase inhibitor), histatin 3 increased the binding between those proteins but not with a peptide capable of binding to HSC70. Moreover histatin 3 prevented ATP-dependent dissociation of HSC70-p27(Kip1). ATP was unable to form a histatin 3-HSC70(D10N)-p27(Kip1) complex (HSC70(D10N) is a mutant attenuating ATPase activity). These findings suggest that histatin 3 may be involved in cell proliferation through the regulation of HSC70 and p27(Kip1) in oral cells.

Transcriptional regulation of the salivary histatin gene: finding of a strong positive regulatory element and its binding protein.

Histatins are salivary proteins found and expressed in human salivary glands. They play a role in the non-immune system of antimicrobial defense, for instance, against Candida albicans. The transcriptional regulatory sequences of the histatin gene, HIS1, have remained obscure for a long time. Here, we cloned the putative promoter from human genomic DNA and tested it in a luciferase reporter system. This promoter is much more active in salivary gland cells than in other cell types. Analysis of deletion mutants revealed that the region encompassing -2254 to -1748 is a strong positive transcriptional element, and its functional core sequence (termed HTN27 box) works in correct and reverse orientations in synergy with downstream sequences, the region spanning -680 to +28 and a proximal promoter. The plus single-stranded HTN27 box is specifically bound by a 100 kDa protein that is present in HSG cells, but not in HeLa cells. These findings indicate that the regulation of the histatin gene expression may be intricate, and it seems to have a cell-type preference in the salivary gland cells.

Polymorphism of genes encoding toll-like receptors and inflammatory cytokines in periodontal disease in the Japanese population.

Periodontitis, which is a widespread and major dental disease, is a multifactorial, lifestyle-related disease and has been analyzed for gene polymorphism. We examined the frequency of the polymorphisms of the pro-inflammatory cytokine genes IL-1 A (-889) and IL-1 B (+3953) in relation to periodontitis in the Japanese population. We also examined whether polymorphism of TLR2 (Arg677Trp) and TLR4 (Asp299Gly), which are receptors recognized by periodontopathic bacteria, may also be associated with periodontitis. The subjects were 92 Japanese individuals, among whom 43 had periodontitis and 49 were healthy controls. We isolated genomic DNA from lingual mucosal cells and tested them for single nucleotide polymorphisms by polymerase chain reaction-restriction fragment length polymorphism. The incidence of polymorphisms was analyzed statistically by Fisher's exact test, and the sensitivity and specificity of the gene polymorphisms were calculated. The purpose was to determine whether such polymorphisms might be effectively used in the diagnosis of periodontitis. However, we found no evidence that the gene polymorphism of IL-1A (p = 0.082), IL-1 B (p = 0.180), TLR2 (p = 1.000) or TLR4 (p = 1.000) and overall gene polymorphism in any of the genes (p = 0.752) correlate with periodontitis. The sensitivity (14.0%) and specificity (83.7%) of the mutations found in all of the genes were low. Therefore, we advise against using the analyses of polymorphism of these genes to detect periodontitis in the Japanese population.

Recovery of genomic DNA from lingual mucosal cells in sufficient quantity and quality.

PURPOSE: To investigate whether genomic DNA can be purified in sufficient quantity and quality from the oral cavity. METHODS: One milliliter of peripheral blood and saliva were collected. The buccal and lingual mucosal cells were also obtained using 10 strokes with a swab or a toothbrush, respectively. All materials were centrifuged and the cells were lysed by adding sodium dodecyl-sulfate and proteinase K. The DNAs were extracted with phenol and precipitated with ethanol followed by electrophoresing on 0.8% agarose gel. The purified DNAs were digested with restriction enzyme Dpn I and Mbo I, respectively. Amplification of the IL-1A gene by PCR was carried out using the purified DNAs and electrophoresing on polyacrylamide gel. RESULTS: DNA was obtained from lingual mucosal cells collected with a toothbrush. Only about one-thirtieth of the recovered DNA was of non-human origin (bacterial contaminants from the oral cavity). Judging from the PCR amplifications of the IL-1A gene, the DNA extracted from lingual cells was of sufficient quality, in all respects indistinguishable from the DNAs extracted from the other specimen, such as peripheral blood, saliva and buccal mucosal cells collected with a swab, and in sufficient quantity. Our results indicate that it is possible to purify DNAs from lingual mucosal cells collected with a toothbrush in a simple and safe manner. Compared to DNA samples from patients by blood extraction, the described method also had the advantage of being painless and not inducing mental distress.