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Abstract Background Whereas the antimicrobial peptides hBD-2 and -3 are related to inflammation, the constitutively expressed hBD-1 might function as 8p tumour suppressor gene and thus play a key role in control of transcription and induction of apoptosis in malignant epithelial tumours. Therefore this study was conducted to characterise proteins involved in cell cycle control and host defence in different benign and malignant salivary gland tumours in comparison with healthy salivary gland tissue. Methods 21 paraffin-embedded tissue samples of benign (n = 7), and malignant (n = 7) salivary gland tumours as well as healthy (n = 7) salivary glands were examined immunohistochemically for the expression of p53, bcl-2, and hBD-1, -2, -3. Results HBD-1 was distributed in the cytoplasm of healthy salivary glands and benign salivary gland tumours but seems to migrate into the nucleus of malignant salivary gland tumours. Pleomorphic adenomas showed cytoplasmic as well as weak nuclear hBD-1 staining. Conclusion HBD-1, 2 and 3 are traceable in healthy salivary gland tissue as well as in benign and malignant salivary gland tumours. As hBD-1 is shifted from the cytoplasm to the nucleus in malignant salivary gland tumours, we hypothesize that it might play a role in the oncogenesis of these tumours. In pleomorphic adenomas hBD-1 might be connected to their biologic behaviour of recurrence and malignant transformation.

Periodontitis is a common complex inflammatory disease of the oral cavity. It is characterized by inflammation of gingival tissues and alveolar bone loss. Recently, a genome-wide association study and 2 genome-wide association study meta-analyses found 2 associated regions (haplotype blocks) at the inhibitory immune receptor gene SIGLEC5 to increase the risk for periodontitis. The aims of the current study were the identification of the putative causal variants underlying these associations, characterization of their molecular biological effects, and validation of SIGLEC5 as the target gene. We mapped the associated single-nucleotide polymorphisms to DNA elements with predictive features of regulatory functions and screened the associated alleles for transcription factor (TF) binding sites. Antibody electrophoretic mobility shift assays (EMSAs) with allele-specific probes were used to identify TF binding and to quantify allele-specific effects on binding affinities. Luciferase reporter assays were used to quantify the effect directions and allele-specific strength of the associated regulatory elements. We used CRISPR-dCas9 gene activation to validate SIGLEC5 as a target of the association. EMSA in peripheral blood mononuclear cells showed that E-26 transformation-specific TF-related gene (ERG) binds at rs11084095, with almost complete loss of binding at the minor A-allele. Allele-specific reporter genes showed enhancer function of the DNA sequence at rs11084095, which was abrogated in the background of the A-allele. EMSA in B lymphocytes showed that TF MAF bZIP (MAFB) binds at the common G-allele of rs4284742, whereas the minor A-allele reduced TF binding by 69%, corresponding to 9-fold reduction of luciferase reporter gene activity by the A-allele. Using CRISPR-dCas9, we showed that the enhancer at rs4284742 strongly activated SIGLEC5 expression, validating this gene as the target gene of the association. We conclude that rs11084095 and rs4284742 are putatively causal for the genome-wide significant associations with periodontitis at SIGLEC5 that impair ERG and MAFB binding, respectively. [ABSTRACT FROM AUTHOR]

Periodontitis is characterized by alveolar bone loss leading to tooth loss. A small proportion of patients develop severe periodontitis at the juvenile or adolescent age without exposure to the main risk factors of the disease. It is considered that these cases carry rare variants with large causal effects, but the specific variants are largely unknown. In this study, we performed exome sequencing of 5 families with children who developed stage IV, grade C, periodontitis between 3 and 18 y of age. In 1 family, we found compound heterozygous variants in the gene CTSC (p.R272H, p.G139R), 1 of which was previously identified in a family with prepubertal periodontitis. Subsequent targeted resequencing of the CTSC gene in 24 patients <25 y of age (stage IV, grade C) identified the known mutation p.I453V (odds ratio = 4.06, 95% CI = 1.6 to 10.3, P = 0.001), which was previously reported to increase the risk for adolescent periodontitis. An affected sibling of another family carried a homozygous deleterious mutation in the gene TUT7 (p.R560Q, CADD score >30 [Combined Annotation Dependent Depletion]), which is implicated in regulation of interleukin 6 expression. Two other affected siblings shared heterozygous deleterious mutations in the interacting genes PADI1 and FLG (both CADD = 36), which contribute to the integrity of the environment–tissue barrier interface. Additionally, we found predicted deleterious mutations in the periodontitis risk genes ABCA1, GLT6D1, and SIGLEC5. We conclude that the CTSC variants p.R272H and p.I453V have different expressivity and diagnostic relevance for prepubertal and adolescent periodontitis, respectively. We propose additional causal variants for early-onset periodontitis, which also locate within genes that carry known susceptibility variants for common forms. However, the genetic architecture of juvenile periodontitis is complex and differs among the affected siblings of the sequenced families. [ABSTRACT FROM AUTHOR]

Genome-wide association studies identified various loci associated with periodontal diseases, but assigning causal alleles remains difficult. Likewise, the generation of biological meaning underlying a statistical association has been challenging. Here, we characterized the genetic association at the gene ST8SIA1 that increases the risk for severe periodontitis in smokers. We used CRISPR/dCas9 activation and RNA-sequencing to identify genetic interaction partners of ST8SIA1 and to determine its function in the cell. We used reporter gene assays to identify regulatory elements at the associated single-nucleotide polymorphisms (SNPs) and to determine effect directions and allele-specific changes of enhancer activity. Antibody electrophoretic mobility shift assays proved allele-specific transcription factor binding at the putative causal SNPs. We found the reported periodontitis risk gene ABCA1 as the top upregulated gene following ST8SIA1 activation. Gene set enrichment analysis showed highest effects on integrin cell surface interactions (area under the curve [AUC] = 0.85; q = 4.9 × 10−6) and cell cycle regulation (AUC = 0.89; q = 1.6 × 10−5). We identified 2 associated repressor elements in the introns of ST8SIA1 that bind the transcriptional repressor BACH1. The putative causative variant rs2012722 decreased BACH1 binding by 40%. We also pinpointed ST8SIA1 as the target gene of the association. ST8SIA1 inhibits cell adhesion with extracellular matrix proteins, integrins, and cell cycle, as well as enhances apoptosis. Likewise, tobacco smoke reportedly results in an inhibition of cell adhesion and a decrease in integrin-positive cells and cell growth. We conclude that impaired ST8SIA1 repression, independently caused by reduced BACH1 binding at the effect T allele, as well as by tobacco smoke, contributes to higher ST8SIA1 levels, and in smokers who carry the effect T allele, both factors would be additive with damaging effects on the gingival barrier integrity. The activity of ST8SIA1 is also linked with the periodontitis risk gene ABCA1. [ABSTRACT FROM AUTHOR]

A metagenomics analysis showed a strongly increased frequency of the protozoan Entamoeba gingivalis in inflamed periodontal pockets, where it contributed the second-most abundant rRNA after human rRNA. This observation and the close biological relationship to Entamoeba histolytica, which causes inflammation and tissue destruction in the colon of predisposed individuals, raised our concern about its putative role in the pathogenesis of periodontitis. Histochemical staining of gingival epithelium inflamed from generalized severe chronic periodontitis visualized the presence of E. gingivalis in conjunction with abundant neutrophils. We showed that on disruption of the epithelial barrier, E. gingivalis invaded gingival tissue, where it moved and fed on host cells. We validated the frequency of E. gingivalis in 158 patients with periodontitis and healthy controls by polymerase chain reaction and microscopy. In the cases, we detected the parasite in 77% of inflamed periodontal sites and 22% of healthy sites; 15% of healthy oral cavities were colonized by E. gingivalis. In primary gingival epithelial cells, we demonstrated by quantitative real-time polymerase chain reaction that infection with E. gingivalis but not with the oral bacterial pathogen Porphyromonas gingivalis strongly upregulated the inflammatory cytokine IL8 (1,900 fold, P = 2 × 10-4) and the epithelial barrier gene MUC21 (8-fold, P = 7 × 10-4). In gingival fibroblasts, we showed upregulation of the collagenase MMP13 (11-fold, P = 3 × 10-4). Direct contact of E. gingivalis to gingival epithelial cells inhibited cell proliferation. We indicated the strong virulence potential of E. gingivalis and showed that the mechanisms of tissue invasion and destruction are similar to the colonic protozoan parasite E. histolytica. In conjunction with abundant colonization of inflamed periodontal sites and the known resistance of Entamoeba species to neutrophils, antimicrobial peptides, and various antibiotics, our results raise the awareness of this protozoan as a potential and, to date, underrated microbial driver of destructive forms of periodontitis. [ABSTRACT FROM AUTHOR]