Construction and characterization of a multilayered gingival keratinocyte culture model : the TURK-U model

In construction of epithelial cells as multilayers, the cells are grown submerged to confluence on fibroblast-embedded collagen gels and, then, lifted to air to promote their stratification. We recently demonstrated that gingival epithelial cells form uniform monolayers on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium, which allows the cells to grow at an air-liquid-solid interface from the beginning of the culturing protocol. In this study, the aim was to further develop our previous model to form a multilayered gingival epithelial culture model. Two different epithelial cell lines (HaCaT from skin and HMK from gingiva) were used in all experiments. Both cell lines were grown first as monolayers for 3 days. After that, keratinocytes were trypsinized, counted and seeded on a sterile semi-permeable nitrocellulose membrane placed on the top of a semi-solid growth medium, forming an air-liquid-solid interface for the cells to grow. At days 1, 4, and 7, epithelial cells were fixed, embedded in paraffin, and sectioned for routine Hematoxylin-Eosin staining and immunohistochemistry for cytokeratin (Ck). At day 1, HMK cells grew as monolayers, while HaCaT cells stratified forming an epithelium with two to three layers. At day 4, a stratified epithelium in the HMK model had four to five layers and its proliferation continued up to day 7. HaCaT cells formed a dense and weakly proliferating epithelium with three to four layers of stratification at day 4 but the proliferation disappeared at day 7. At all days, both models were strongly positive for Ck5, Ck7, and Ck 19, and weakly positive for Ck10. Gingival epithelial cells stratify successfully on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium. This technique allows researchers to construct uniform gingival epithelial cell multilayers at an air-liquid-solid interface, without using collagen gels, resulting in a more reproducible method.Peer reviewe

Cumulative use of salivary markers with an adaptive design improves detection of periodontal disease over fixed biomarker thresholds

Objective: Aim was to analyze the diagnostic ability of cumulative risk score (CRS), which uses salivary levels of Porphyromonas gingivalis, interleukin (IL)-1 beta, and matrix metalloproteinase (MMP)-8 in an adaptive design, compared to previously reported thresholds of each marker alone. Materials and Methods: Oral and general health information of 463 participants were included in the analysis. Having the percentage of bleeding on probing (BOP) > 25%, having at least two sites with probing pocket depth (PPD) of 4-5 mm or having at least one tooth with alveolar bone loss (ABL) of at least 1/3 of the root length were accepted as outcome variables. Being above the salivary threshold concentrations of P. gingivalis, IL-1 beta, and MMP-8 and CRS values were used as explanatory variables. Receiver operating characteristics (ROC) producing an area under the curve (AUC) and multinomial regression analysis were used in statistical analysis. Results: CRS provided AUCs larger than any other tested biomarker threshold. Sensitivity and specificity of CRS for detecting clinical markers of periodontitis were acceptable, and a strong association was observed between the highest CRS score and having at least two sites with PPD of 4-5 mm. Conclusion: CRS brings additional power over fixed thresholds of single biomarkers in detecting periodontitis.Peer reviewe

Hormone Therapy Failure in Human Prostate Cancer: Analysis by Complementary DNA andTissue Microarrays

BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P = .0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P = .0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cance

Use of Saliva in Diagnosis of Periodontitis: Cumulative Use of Bacterial and Host-Derived Biomarkers

Periodontitis is an infection-induced inflammatory disease of the tooth supporting tissues. Treatment of periodontal diseases and regeneration of the effected tissues can be possible only in the early diagnosis of the disease. If left undiagnosed or untreated, periodontitis leads to irreversible soft and hard tissue destruction and finally to tooth loss. Saliva is known to contain inflammatory mediators, host tissue and cell degradation products as well as microbial metabolites and enzymes, reflecting the health status of the oral cavity. In this topic, in collaboration with the well-known scientists working on the field of salivary diagnostics, we demonstrate evidence on monitoring periodontitis by salivary analysis

Prevalence of periodontal bacteria in saliva of Kuwaiti children at different age groups

Summary: Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, Tannerella forsythensis and Porphyromonas gingivalis and to a lesser extent Prevotella intermedia and Prevotella nigrescens, are Gram-negative species that are associated with destructive periodontitis. Studies from different parts of the world have shown variable detection rates of periodontal organisms. Hardly any data exist on their carriage in children living in the Middle East. This study was designed to determine the detection of these species in the oral cavity of 240 generally healthy Kuwaiti children, divided into five age groups: <6 years (n = 40), 6–9 years (n = 60), 10–12 years (n = 40), 13–15 years (n = 40) and 16–18 years (n = 60). Saliva was used as the microbiological specimen, and the samples were analyzed by molecular methods using multiplex PCR. A total of 185 (77.1%) of the 240 children were colonized by at least one of the target periodontal bacteria. In all age groups, P. nigrescens was the most prominent and detected in saliva of 15%, 32%, 63%, 50%, and 47% of the children at the five age groups, respectively. P. gingivalis was detected only occasionally. Only few pathogens were found before the permanent dentition, i.e. at the age of <6 years. The highest carriage rates were from the groups between 6 and 15 years of age. The salivary carriage of the pathogens was essentially similar in the age groups of 10–12 years and 13–15 years. In conclusion, except for P. gingivalis, the examined periodontal pathogens are relatively common findings in Kuwaiti children and colonize the oral cavity from childhood onwards. Keywords: Prevalence, Periondontopathic pathogens, Children, Kuwai

Human neutrophil peptide-1 affects matrix metalloproteinase-2,-8 and-9 secretions of oral squamous cell carcinoma cell lines in vitro

Objectives: The present study aimed to investigate the effect of HNP-1 on the matrix metalloproteinase (MMP)-2, -8 and -9 secretions of two oral squamous cell carcinoma (OSCC) cell lines (UT-SCC-43A and UT-SCC-43B). Design: In all experiments, the two OSCC cell lines were incubated with graded concentrations (0,1, 5, and 10 mu g/ml) of HNP-1 for 24 and 48 h. Cell viability was measured using a colorimetric proliferation test and cell death was analyzed with a colorimetric cytotoxicity detection kit. Enzyme activity of MMP-2 and MMP-9 was detected by using gelatin zymography, and molecular weight forms of MMP-8 were determined by Western-blot and a densitometric quantitation method. Results: Both cell lines showed a significant increase in LDH toxicity at 24 h (UT-SCC-43A: p = 0.005 & UT-SCC-43B: p = 0.014). Reduced gelatinolytic activities of proMMP-2 were detected in UT-SCC-43B cell line after 24 and 48 h of incubation with HNP-1 (1 mu g/ml: p <0.001, 5 mu g/ml: p <0.001, and 1011g/ml: p = 0.0225). MMP-8 levels of both cell lines decreased at 200-250 kDa after 24 h of incubation, while after 48 h only UT-SCC-43B decreased at 45-50 kDa. Conclusions: Our results indicate that HNP-1 suppresses the secretion of MMP-2,-8, and -9 in OSCC cell lines. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe