High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview.

In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.

Clinical Evaluation of Diode (980 nm) Laser-Assisted Nonsurgical Periodontal Pocket Therapy: A Randomized Comparative Clinical Trial and Bacteriological Study.

Background: Mechanical debridement is the gold standard in the periodontitis therapy. However, it is suggested that adjunctive use of lasers can result in a more effective treatment outcome. Objective: Evaluate the efficiency of diode laser-assisted nonsurgical therapy of periodontitis as adjunctive to scaling and root planing (SRP). Methods: One hundred sixty vertical bone defects [pocket depth (PD) at baseline ≥6 mm] had been randomly allocated to receive SRP alone (group C) or SRP coupled to a diode laser (980 nm) protocol (group C+L): SRP, irrigation with hydrogen peroxide solution (3%), de-epithelization of the internal and external gingiva followed by blood stabilization, and coagulation by laser beam were made. Beam parameters: 10 µsec/pulse duration, 10 kHz, pick power of 10 W, average power of 1 W, and fiber diameter of 400 µm. Plaque index (PI), bleeding on probing, gingival recession (GR), clinical attachment level (CAL), and PD were measured at baseline, at 6 weeks, 12 weeks, 18 weeks, 6 months, and 12 months. Microbiological data were collected randomly from 26 pockets from both groups at baseline, 6 weeks, 12 weeks, and 6 months after treatment. Results: At all periods of follow-up, there was a significant difference between both groups in all clinical parameters except in GR. In group C+L, 76% of pockets had PD ≤3 mm after 12 months of follow-up and an average of PD = 1.77 ± 0.46 mm, while 56% of pockets in group control (C) had an average of PD = 5.00 ± 0.83 mm after 12 months of follow-up. Total bacteria count in group C + L was significantly lower compared to group C only at 12 weeks and 6 months of follow-up. Furthermore, there was high significant decrease in the number of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia at all the follow-up periods. Conclusions: As adjunctive to SRP, diode laser-assisted nonsurgical therapy of periodontitis has significantly improved clinical parameters of PI and POB and has significantly reduced the clinical attachment loss (CAL) and PD compared to the control group after 1 year of follow-up. A significant reduction in periodontal pathogens has been observed in group C + L only at 12 weeks and 6 months of follow-up.

A Randomized Comparative Clinical Study to Evaluate the Longevity of Esthetic Results of Gingival Melanin Depigmentation Treatment Using Different Laser Wavelengths (Diode, CO2, and Er:YAG).

Background: Gingival melanin hyperpigmentation is due to excessive deposition of melanin granules. The duration of pigmentation reappearance after treatment using different laser wavelengths remains controversial. Objective: The study aims to assess the longevity of gingival depigmentation (GD) and the consistency in esthetic results as three laser wavelengths (Er:YAG laser, CO2 laser, and diode laser, 980 nm) were used in two different groups (smokers and nonsmokers). This is attained by comparing the periods of time in each group before pigmentation reappearance. Methods: Seventy-two subjects were divided into daily smokers (S) and nonsmokers. Subjects underwent a randomized GD with: Erbium laser (Er), CO2 laser (CO2), and Diode laser (Diode). The subjects were divided into six groups: S and nonsmokers were treated with three different wavelengths. Irradiation was performed until there was no visible pigmentation. For qualitative measurement, Hedin Melanin Index (HMI) was used, before treatment, after 2 weeks, and until 60 months. Pigmentation reappearance of degree 1 or above of the HMI was noted. Descriptive statistics were also calculated. Results: HMI showed a 0 in all groups after 14 days of treatment. The time before pigmentation rebound was: Diode > CO2 > S-Diode > S-CO2 > Er > S-Er. The first signs of relapse shown among all groups were seen in the group S-Er group. The longest time before rebound was observed with the Diode group for the nonsmoker. Conclusions: Diode laser provides the longest-term stability in treatment. Smoking negatively affects the longevity of GD. Er laser gives the shortest time before the reappearance of gingival pigmentation.

p53 Overexpression in Oral Mucosa in Relation to Shisha Smoking in Syria and Lebanon

Introduction: Shisha (waterpipe) smoking is becoming a very prevalent form of tobacco consumption in the Middle east and use is growing over the world. Smoking-related malignancies have a high genome-wide burden of mutations, including examples in the gene encoding p53. Aims: To investigate alterations in p53 immunohistochemical expression in the normal, pre-malignant, malignant oral mucosa in relation to Shisha smoking habits. Materials and Methods: A total of 105 paraffin embedded tissue sections of OSCCs (52 smokers,53 non-smokers), 96 of premalignant lesions (48 smokers,48 non-smokers) and 60 normal oral mucosa. Some 30 patients with a history of Shisha smoking daily for more than 5 years were also investigated for mutant expression of p53. Tissue samples were considered positive for p53 staining when any positive cells of epithelial origin could be detected. Results: The majority (74.3%) of oral squamous cell carcinomas showed positive staining for p53 expression (83.1% and 65.5% with Shisha smokers and non-smokers, respectively). In the 96 premalignant lesions, about 23% from non-smokers and 41.7% from smokers showed p53 positivity. In normal epithelium, P53 positive cells were noted in 6.6% of non-smokers and 16.6% of smokers. Positive correlations with Shisha smoking were evident for the following groups: WDOSCC, MDOSCC, mild dysplasia G1, moderate dysplasia G2 and in normal mucosa using Student's t- test, P value<0.05. Conclusion: These results strongly suggest that p53 mutations are associated with Shisha smoking in OSCC, pre-malignant lesions and normal mucosa of the oral cavity.

Transmission of Induced Chromosomal Aberrations through Successive Mitotic Divisions in Human Lymphocytes after In Vitro and In Vivo Radiation.

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

CCR7 and CXCR4 Expression in Primary Head and Neck Squamous Cell Carcinomas and Nodal Metastases ­ a Clinical and Immunohistochemical Study

Background: Squamous cell carcinomas (SCCs) are common head and neck malignancies demonstrating lymph node LN involvement. Recently chemokine receptor overxpression has been reported in many cancers. Of particular interest, CCR7 appears to be a strong mediator of LN metastases, while CXCR4 may mediate distant metastases. Any relations between their expression in primary HNSCCs and metastatic lymph nodes need to be clarified. Aims: To investigate CCR7 andCXCR4 expression in primary HNSCCs of all tumor sizes, clinical stages and histological grades, as well as involved lymph nodes, then make comparisons, also with control normal oral epithelium. Materials and Methods: The sample consisted of 60 formalin-fixed, paraffin-embedded specimens of primary HNSCCs, 77 others of metastasi-positive lymph nodes, and 10 of control normal oral epithelial tissues. Sections were conventionally stained with H&E and immunohistochemically with monoclonal anti-CCR7 and monoclonal anti-CXCR4 antibodies. Positive cells were counted under microscopic assessment in four fields (X40) per case. Results: There was no variation among primary HNSCC tumors staining positive for CCR7 and CXCR4 with tumor size of for CCR7 with lymph node involvement. However, a difference was noted between primary HNSCC tumors stained by CXCR4 with a single as compared to more numerous node involvement. CXCR4 appear to vary with the clinical stagebut no links were noted with histological grades. Staining for primary HNSCC tumors and metastatic lymph nodes correlated.

Detection and automated scoring of dicentric chromosomes in nonstimulated lymphocyte prematurely condensed chromosomes after telomere and centromere staining.

PURPOSE: To combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose-response curve and automation of the process. METHODS AND MATERIALS: Blood samples from healthy donors were exposed to (60)Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory. RESULTS: We successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose-response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings. CONCLUSION: The introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw.