The influence of the tooth preparation finish line position on the expression of matrix metalloproteinase-9 and the presence of periodontopathogens in the gingival crevicular fluid.

OBJECTIVE: The objective of the study was to determine the concentration of matrix metalloproteinase 9 (MMP-9) and changes in the presence of periodontopathogens in the gingival crevicular fluid before and after tooth preparation with the subgingival and equigingival finish line position. PATIENTS AND METHODS: The clinical prospective study included 20 subjects with an indication for upper canine preparation, with the subgingival (group 1) and equigingival finish line (group 2). Samples were taken in four observation intervals: 5 minutes before (control samples), as well as 15 minutes, 24 and 72 hours after tooth preparation (experimental samples). Measurement of MMP-9 was done using Enzyme-linked Immunosorbent Assay (ELISA). The presence of bacteria in the gingival fluid was proven by the Polymerase chain reaction (PCR) analysis. RESULTS: The MMP-9 values did not differ statistically significantly between the groups (p=0.524). The MMP-9 values showed a statistically significant difference in the given observation period (p<0.001) with a significant linear increase in values (p<0.001). A significant quadratic trend recorded a decrease in the MMP-9 values 15 minutes after preparation, and an increase 24 hours after preparation, without a significant difference in the interaction between groups (p=0.392). After preparation, a significant difference in the presence of periodontopathogens was confirmed, i.e., a decrease in the presence of Prevotella intermedia (p=0.025) and Tannerella forsythia (p=0.016) in group 1, and an increase in the presence of Aggregatibacter actinomycetemcomitans in both groups (p=0.029, p=0.026). CONCLUSIONS: The study is a good basis for determining the influence of tooth preparation on gingival inflammation, with therapeutic (choice of preparation technique) and preventive significance regarding the protection of the periodontal tissue from possible iatrogenic damage.

Somatic genomic imbalances in 'tumour-free' surgical margins of oral cancer.

Up to 30% of oral squamous cell carcinoma (OSCC) patients develop local recurrence and distant metastasis. The molecular status of histologically cancer-free tumour margins could be a critical factor in predicting tumour behaviour. The aim of this study was to detect somatic genomic imbalances in OSCC with emphasis on the surgical margins. DNA was isolated from tumour tissues, margin tissues, and blood samples (used as control) obtained from 11 OSCC patients, and genome-wide array comparative genomic hybridization was performed. Imbalances were present in both tumours and margins, although, as expected, they were more prevalent in tumours (duplications, P = 0.0002; deletions, P = 0.0001). Duplications were more frequent than deletions in both tumours and margins, but without statistical significance. Fifteen imbalances in tumour tissues were recurrent and all of them were duplications. Four of these were found both in tumours and margins and involved chromosomes 1q, 8p, Xp, Yp, and Yq. Four imbalances were recurrent in margin tissue and all of them were duplications (autosomes 8 and 17 and both sex chromosomes). Histologically 'cancer-free' margins hide genomic alterations consistent with unexplained OSCC recurrences. Establishing the molecular status of the margins could improve outcome prediction.

Association of polymorphisms in TNF-α, IL-1ß, GSTM and GSTT genes with apical periodontitis: is there a link with herpesviral infection?

AIM: To investigate the possible association between TNFα (-308 G/A) and IL-1ß (-511 C/T) single nucleotide polymorphisms (SNPs) and GSTT and GSTM deletion polymorphisms and risk of apical periodontitis (AP) development, and determine the association of different genotypes with the presence of herpesviral infection in AP. METHODOLOGY: The study included 120 periapical lesions and 200 control samples. Gene polymorphism analysis was performed using either polymerase chain reaction (PCR) or PCR/ restriction fragment length polymorphism (RFLP). Relative gene expression of TNF-α and IL-1ß was analysed using reverse transcriptase - real-time PCR. The presence of Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) was assessed by nested PCR. Chi-square and Fisher's exact tests and logistic regression analyses were done for polymorphisms, whilst Mann-Whitney U-test was performed for the expression analysis. The expected frequency of variants was analysed by the Hardy-Weinberg equilibrium test. RESULTS: TNF-α (-308 G/A) SNP increased AP susceptibility for heterozygous (odds ratio (OR) = 1.72, 95% confidence interval (CI) = 1.06-2.80, P = 0.027) and homozygous (OR = 8.55, 95% CI = 1.77-41.36, P < 0.001) carriers of the variant A allele. On the other hand, IL-1ß (-511 C/T) polymorphism exerted a protective effect both in heterozygotes (OR = 0.540, 95% CI = 0.332-0.880, P = 0.013) and homozygotes (OR = 0.114, 95% CI = 0.026-0.501, P < 0.001). In addition, GSTM1 and GSTT1 null genotypes separately, as well as concomitantly, were associated with an increased risk for AP development (P < 0.001). The null GSTT1 genotype increased approximately twice the risk of Epstein-Barr infection (EBV) in AP (OR = 2.17, 95% CI = 1-4.71, P = 0.048), whilst TNF-α SNP decreased it, both in heterozygotes (OR = 0.20, 95% CI = 0.08-0.48, P < 0.001) and AA homozygotes (OR = 0.07, 95% CI = 0.01-0.37, P = 0.001). CONCLUSIONS: GSTM and GSTT deletion polymorphisms, as well as TNFα (-308 G/A) SNP, are associated with increased risk, whereas IL-1ß (-511 C/T) polymorphism decreases the risk of AP development. GSTT and TNFα polymorphisms also appear to modulate the risk of EBV infection in Serbian patients with AP.

Genetic and epigenetic alterations in the tumour, tumour margins, and normal buccal mucosa of patients with oral cancer.

Despite adequate surgical resection, oral squamous cell carcinoma (OSCC) shows a high rate of recurrence and metastasis, which could be explained by the presence of molecular alterations in seemingly normal tumour margins and the entire oral mucosa. The aims of this study were (1) to assess the presence of gene amplification (c-Myc and HER2) and promoter methylation (p14 and p16) in the tumours, tumour margins, and unaffected oral mucosa of 40 OSCC patients, and (2) to evaluate the possibility of using these alterations as prognostic markers. c-Myc and HER2 genes were quantified by means of real-time PCR (qPCR), and p14 and p16 methylation status was determined by methylation-specific PCR (MSP PCR). All tissues examined exhibited molecular alterations in various proportions. Tumour tissues, as expected, showed the highest prevalence of alterations, while oral mucosa showed the lowest. Multiple alterations (co-alterations) in tumours and tumour margins were significantly more frequent than in unaffected oral mucosa (P<0.001 and P=0.027, respectively). HER2 amplification in margin tissue (P<0.001) and swabs (P=0.013), as well as the existence of three co-alterations in margins (P=0.001) and macroscopically unaffected oral mucosa (P<0.001) were correlated with shorter disease-specific survival.

Levels of oxidative stress biomarkers and bone resorption regulators in apical periodontitis lesions infected by Epstein-Barr virus.

AIM: To investigate whether apical periodontitis lesions infected by Epstein-Barr virus (EBV) exhibit higher levels of oxidative stress biomarkers [8-hydroxydeoxyguanosine (8-OHdG) and oxidized glutathione (GSSG)] and bone resorption regulators [receptor activator of nuclear factor (NF-κB) ligand (RANKL) and osteoprotegerin (OPG)] compared to EBV-negative periapical lesions and healthy pulp tissues. METHODOLOGY: The experimental group consisted of 30 EBV-positive and 30 EBV-negative periapical lesions collected in conjunction with apicoectomy. The pulp tissues of 20 impacted third molars were used as healthy controls. The qualitative and quantitative analysis of EBV was performed by nested and real-time polymerase chain reaction (PCR), respectively. The levels of RANKL and OPG were analysed by reverse transcriptase real-time PCR. The levels of 8-OHdG and GSSG were determined by enzyme-linked immunosorbent assay (ELISA). Mann-Whitney U-test and Spearman's correlation were used for statistical analysis. RESULTS: The levels of RANKL, OPG, 8-OHdG and GSSG were significantly higher in apical periodontitis lesions compared to healthy pulp controls (P = 0.001, P < 0.001, P < 0.001 and P < 0.05, respectively). RANKL and OPG mRNA expression was significantly higher in EBV-positive compared to EBV-negative periapical lesions (P < 0.05). There was no significant correlation between EBV copy numbers and levels of RANKL, OPG, 8OH-dG and GSSG in apical periodontitis. CONCLUSION: Levels of bone resorption regulators and oxidative stress biomarkers were increased in apical periodontitis compared to healthy pulp tissues. EBV-positive periapical lesions exhibited higher levels of RANKL and OPG compared to EBV-negative periapical lesions. EBV may contribute to progression of apical periodontitis via enhanced production of bone resorption regulators.

Herpesviruses viral loads and levels of proinflammatory cytokines in apical periodontitis.

OBJECTIVES: This study aimed to analyse Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) viral loads in symptomatic and asymptomatic apical periodontitis lesions, to determine levels of TNF-α, IL-1ß and IL-6 in these lesions and to investigate a possible correlation between herpesviral copy numbers and levels of proinflammatory cytokines. MATERIALS AND METHODS: A total of 100 samples of apical periodontitis were subjected to HCMV and EBV copy numbers analysis by nested polymerase chain reaction (PCR) and TaqMan real-time PCR. The concentrations of TNF-α, IL-1ß and IL-6 were determined by ELISA method. SPSS software was used for statistical analysis. RESULTS: There were no significant differences in the occurrence of EBV and HCMV between symptomatic and asymptomatic periapical lesions (p = .686, p = .879, respectively). Only 12 of 74 EBV (16.2%) and four of 54 HCMV (13.5%) nested PCR-positive samples showed increased viral copy numbers above the limit of 125 copies/ml. There was no significant correlation between the levels of analysed proinflammatory cytokines and herpesviral copy numbers in our sample. CONCLUSION: The observed low viral loads point to a relatively rare occurrence of active EBV and HCMV infection in our sample. Latent herpesviral infection does not enhance the production of investigated proinflammatory cytokines.

Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise - possibilities and limitations.

The beneficial health-related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.

Persistent Organic Pollutants in albacore tuna (Thunnus alalunga) from Reunion Island (Southwest Indian Ocean) and South Africa in relation to biological and trophic characteristics.

The contamination of albacore tuna (Thunnus alalunga) by Persistent Organic Pollutants (POPs), namely polychlorinated biphenyls (PCBs) and dichlorodiphenyl-trichloroethane (DDT), was investigated in individuals collected from Reunion Island (RI) and South Africa's (SA) southern coastlines in 2013, in relation to biological parameters and feeding ecology. The results showed lower PCB and DDT concentrations than those previously reported in various tuna species worldwide. A predominance of DDTs over PCBs was revealed, reflecting continuing inputs of DDT. Tuna collected from SA exhibited higher contamination levels than those from RI, related to higher dietary inputs and higher total lipid content. Greater variability in contamination levels and profiles was identified in tuna from RI, explained by a higher diversity of prey and more individualistic foraging behaviour. PCB and DDT contamination levels and profiles varied significantly in tuna from the two investigated areas, probably reflecting exposure to different sources of contamination.

Magnesium and iron deficiencies alter Cd accumulation in Salix viminalis L.

Evidence exists that Cd and certain nutrient elements, such as Fe and Mg, could share similar mechanisms of plant uptake and accumulation. Here we report that Mg and Fe deficiency in mature plants of Salix viminalis, grown in hydroponic solutions containing 5 µg ml(-1) of Cd, caused a significant increase in Cd accumulation in roots, stems and leaves. Cd (µg g(-1) dry weight) was determined following three treatments: 1) Cd treatment in complete nutrient solution; 2) Cd treatment with Fe deficiency; and 3) Cd treatment with Mg deficiency, yielding, respectively: in young leaves (65.3, 76.1, and 92.2), mature leaves (51.5 to 76.3 and 87.1), upper stems (80.6, 116.8, and 130.6) lower stems (67.2, 119, and 102.3), roots (377.1, 744.8, and 442,5). Our results suggest that Cd utilizes the same uptake and transport pathways as Mg and Fe. Evidence exists that Mg and Fe uptake and translocation could be further facilitated by plants as an adaptive response to deficiency of these elements. Such physiological reaction could additionally stimulate Cd accumulation. Although Cd uptake was mostly confined in roots, high Cd content in aerial plant parts (51.5-130.6 µg g(-1)) indicates that the analysed Salix viminalis genotype is suitable for phytoextraction.

The amplification of c-erb-B2 in cancer-free surgical margins is a predictor of poor outcome in oral squamous cell carcinoma.

The tumour subtype, TNM classification, and histopathological data are sometimes not sufficient for understanding and assessing the behaviour of oral cancers. In an attempt to find additional markers of tumour biology and behaviour, this study sought to determine the incidence and consequently the relevance of c-erb-B2, c-Myc, and H-ras gene alterations in tumour-free margins of oral squamous cell carcinoma (OSCC). Fifty samples of OSCC were analyzed for c-erb-B2 and c-Myc amplification by real-time polymerase chain reaction and for H-ras point mutations by sequencing. A relatively high incidence of genetic lesions was detected: 22% of cases had c-erb-B2 and 30% had c-Myc amplification, whilst only 12% harboured H-ras mutations. Kaplan-Meier analysis and the log-rank test showed statistically significant differences in 5-year survival rates and relapse between patients with tumour margins positive for c-erb-B2 amplification and those with margins that were negative (P=0.002). H-ras and c-Myc alterations could not be associated with tumour behaviour. Molecular analysis of margins, targeting cancer genes, could identify additional, independent predictors of risk and outcome in OSCC.

Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations.

The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 µM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 µM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 µM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

The utility of 99mTc-EDDA/HYNIC-TOC scintigraphy for assessment of lung lesions in patients with neuroendocrine tumors.

UNLABELLED: Our aim was to assess clinical utility of 99mTc-EDDA/HYNIC-TOC scintigraphy for evaluation of lung lesions in patients with neuroendocrine tumors (NETs). Single photon emission computed tomography (SPECT) of the thorax and whole body scintigraphy were performed in 34 patients using 99mTc-EDDA/HYNIC-TOC. Visual assessment was complemented by semiquantitative evaluation based on tumor to non-tumor (T/NT) ratio. Clinical, laboratory, and histological findings served as the standard for comparison. Enhanced tracer uptake was observed on both SPECT and whole body scintigraphy in 29 of 34 patients (88% sensitivity). T/NT ratios were significantly higher on SPECT than whole body images (2.96+/-1.07 vs.1.70+/-0.43, p KEYWORDS: 99mTc-EDDA/Hynic-TOC, lung involvement of NETs, T/NT ratio.

[Ethanol, water, salt and coma]. / Alkohol, Wasser, Salz und Koma.

We report on a 52-year-old woman with liver cirrhosis who suddenly fell into deep coma after correction of an intercurrent hyponatremia. After exclusion of the commonest causes of coma, the MRI showed a symmetrical osmotic demyelination of both thalami. The newest epidemiological data, the pathophysiology of osmotic demyelination, the threats of the treatment of a prolonged severe hyponatremia as well as the therapeutic options in face of osmotic demyelination are discussed.

A set of 37 microsatellite DNA markers for genetic diversity and structure analysis of Atlantic salmon Salmo salar populations.

Atlantic salmon Salmo salar microsatellite markers from a large database were analysed and selected with technical, economic and genetic criteria to provide an optimized set of polymorphic DNA markers for the analysis of the genetic diversity and the structure of anadromous Atlantic salmon populations. A set of 37 microsatellite markers was identified that are easy to use and provide a high level of differentiation power.

Particle size analysis: 90Y and 99mTc-labelled colloids.

Colloidal particle size is an important characteristic to consider when choosing a radiopharmaceutical for diagnosis and therapeutic purposes in nuclear medicine. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were used to determine the particle-size distribution of (90)Y- and (99m)Tc-labelled antimony trisulfide (Sb(2)S(3)) and tin colloids (Sn-colloid). (90)Y-Sb(2)S(3) and (99m)Tc-Sb(2)S(3) were found to have a diameter of 28.92 +/- 0.14 and 35.61 +/- 0.11 nm, respectively, by PCS. By TEM, (90)Y-Sb(2)S(3) particles were measured to be 14.33 +/- 0.09 nm. (90)Y-labelled Sn colloid were found to exist with a d(v(max1)) of 805 nm and a d(v(max2)) of 2590 nm, by PCS, whereas (99m)Tc-Sn colloid was shown to have more than 80% of radioactive particles of approximately 910 nm by PCS. For (90)Y-labelled Sb(2)S(3) and Sn colloid, a comparison of TEM and PCS indicates that these techniques found significantly different mean diameters. TEM has an excellent resolution necessary for radiocolloid particle-sizing analysis, and it is a desirable size-measuring technique because it is more reliable than PCS.

[Severe behavioral disorder caused by an organic disease]. / Schwere Verhaltensstörung mit organischer Ursache.

A 65-year-old architect was admitted to our Memory Clinic because of rapidly progressive dementia accompanied by behavioral disorders, which severely restricted his functional independence. The physical examination revealed several focal neurological signs, and neuroimaging confirmed a cerebellar hemorrhage, multiple microbleeds and an ischemic lacunar infarct. Executive functioning was highly impaired. This case report illustrates cerebral amyloid angiopathy as an important entity of vascular dementia and highlights the importance of neuroimaging in patients with suspected vascular cognitive disorders.

Detection of gastrointestinal and abdominal infections by 99mTc-ciprofloxacin.

BACKGROUND/AIMS: The aim of the study is detection and evaluation of the abdominal and gastrointestinal infective foci using 99mTc-ciprofloxacin (Laboratory for radioactive isotopes, Vinca). METHODOLOGY: In total 21 patients with clinical suspicion on abdominal or gastrointestinal infection were investigated. In all the patients, planar liver/spleen scintigraphy was performed. Ciprofloxacin chloride (3.5 mg) was mixed with 555 MBq of 99mTc in 3 mL of physiological solution and incubated for 20 min. After slow i.v. injection in a cubital vein, dynamic acquisition (1 f/min) was performed during the first 60 min in the position of interest, followed by static acquisition (500,000 imp) anterior and posterior view, abdomen and pelvis after 1 h and 4 h in all patients. When necessary, additional scintigrams were acquired after 24 h. In all the patients with negative or equivocal findings of planar scintigraphy, emission computerized tomography (SPECT) was performed (60 positions, 6 degrees). Interpretation was made by three independent observers. Additional data were provided using clinical findings, ultrasonography, computed tomography and magnetic resonance imaging, laboratory analyses, and surgical or microbiological confirmation of infection. RESULTS: There were eleven true-positive findings, seven true negative, two were false negative while one was false positive due to intestinal obstruction. Sensitivity was 79%, specificity 91%, positive predictive value 92%, negative predictive value 77%, accuracy 84%. CONCLUSIONS: According to our results, scintigraphy with radiolabeled ciprofloxacin is a useful method for detection and assessment of exact localization of abdominal and gastrointestinal infections.

Subjective assessments of safety, exposure to chemicals and use of personal protection equipment in seafaring.

BACKGROUND: Most of the scientific publications from the maritime area are studies about the mortality and morbidity, while studies about the present hazards of potentially dangerous exposures are relatively rare. AIMS: To describe the seafarers' assessments of the occupational safety on board, their exposure to chemicals and the use of personal protection equipment and to identify the areas for further risk assessment and preventive measures. METHODS: A questionnaire study was carried out in 11 countries among seafarers who attended a regular health examination. RESULTS: The total number of seafarers who participated in the study was 6461 (response rate 93.7%). The occupational safety on board was assessed to be very good or good in 82%. Multivariate analyses showed that the safety was assessed as lowest among ratings, seafarers<30 years of age, work in the engine rooms and on dry cargo ships. It was highest on crude oil tankers and supply ships. Fifty-five per cent of seafarers were exposed to chemicals. Personal protection equipment to chemicals was used 'always/almost always' in 93% of the exposed. Multivariate analysis showed that the use of personal protection was highest on deck, on the largest ships, on roll-on roll-off ships and on crude oil tankers. CONCLUSION: The occupational safety and the use of personal protective equipment was assessed to be significantly different in some strata of the population and in specific working areas and types and sizes of ships.