Quantitative assessment of morphological changes of dental pulp components of teeth affected by occlusal trauma.

AIM: In the general economy of the stomatognathic system functionality, the occlusal function has an important role, meaning both the dental-dental reports and the dynamic reports interarch-interarch. Because of the interrelationships and inter-dependency that govern the functioning of the biological systems, a pathological change affecting a component of the stomatognathic system produces impaired functioning of the others. The aim of the present study is to assess the morphological changes occurred in the dental pulp components of teeth affected by occlusal trauma. MATERIALS AND METHODS: Fragments of dental pulp coming from 45 patients with occlusal trauma were processed using classical histological techniques (formalin fixation and paraffin embedment) and stained with Hematoxylin-Eosin (HE), Masson's trichrome and anti-CD34 antibody immunostaining, in order to highlight the peripheral zone and central connective tissue of dental pulp morphological changes. A set of parameters namely thickness of peripheral zone components, calcifications, fibrosis and vascular density in the dental pulp were assessed individually and based on three topographical criteria namely the affected tooth, the position on the dental arches and the position according to sagittal plane. RESULTS: There was no correlation between morphological changes of dental pulp and the topography of teeth with occlusal trauma. The size of the peripheral area of the dental pulp and that of its components evolved in the same sense, whether it was growth or decrease. Decrease of the peripheral area was associated with the increase of collagen fibers density, calcium deposits and density of the capillary network. The direct correlation between the amount of collagen fibers and vascular density seems somewhat paradoxical but it can be explained by the reemergence of chronic inflammatory events located in the dental pulp. CONCLUSIONS: It seems that dental pulp morphological changes are not influenced by the teeth with occlusal trauma topography. With one exception (the components of peripheral zone), most of the correlations between the dental pulp morphological changes were only suggested but not validated statistically, which requires further studies on larger groups together with the introduction of inflammatory cell population studies.

Morphological changes of gums in occlusal trauma.

AIM: Occlusal trauma causes major modifications of the coverage periodontium, which in turn reflect on the dental unit. The aim of the present study is to evaluate some of the morphological modifications occurred in the marginal periodontium surrounding teeth affected by occlusal trauma. MATERIALS AND METHODS: Fragments of marginal periodontium coming from 51 patients with occlusal trauma were processed using classical histological techniques (formalin fixation and paraffin embedment) and stained with Hematoxylin-Eosin (HE), Masson's trichrome and anti-CD34 antibody immunostaining, in order to highlight the epithelial and connective tissue changes of gingival mucosa. A set of epithelial and connective tissue morphological parameters were assessed individually and based on three topographical criteria concerning the affected tooth. RESULTS: The epithelium and especially its superficial compartment presented changes depending on the tooth type and the dental arch. Epithelial thickness had the tendency to decrease as the fibrosis in both corium compartments and vascular density in the deep corium compartment were increasing. Leukoplakia present around the affected teeth but not always was related with the tooth type and was more obvious as the superficial compartment of the epithelium was thicker and as fibrosis was more reduced in the papillary compartment of the corium. Vascular density reduced when fibrosis process increased in the corium. CONCLUSIONS: Lesions determined by occlusal trauma and their topography can and are influencing locally the different structures of the surrounding periodontium.

Assessing tumor angiogenesis in colorectal cancer by quantitative contrast-enhanced endoscopic ultrasound and molecular and immunohistochemical analysis.

BACKGROUND AND OBJECTIVES: Data on contrast-enhanced endoscopic ultrasound (CE-EUS) for colorectal cancer (CRC) evaluation are scarce. Therefore, we aimed to assess the vascular perfusion pattern in CRC by quantitative CE-EUS and compare it to immunohistochemical and genetic markers of angiogenesis. PATIENTS AND METHODS: We performed a retrospective analysis of CE-EUS examinations of 42 CRC patients, before any therapy. CE-EUS movies were processed using a dedicated software. Ten parameters were automatically generated from the time-intensity curve (TIC) analysis: peak enhancement (PE), rise time (RT), mean transit time, time to peak (TTP), wash-in area under the curve (WiAUC), wash-in rate (WiR), wash-in perfusion index (WiPI), wash-out AUC (WoAUC), and wash-in and wash-out AUC (WiWoAUC). The expression levels of the vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 genes were assessed from biopsy samples harvested during colonoscopy. Microvascular density and vascular area were calculated after CD31 and CD105 immunostaining. RESULTS: Forty-two CE-EUS video sequences were analyzed. We found positive correlations between the parameters PE, WiAUC, WiR, WiPI, WoAUC, WiWoAUC, and N staging (Spearman r = 0.437, r = 0.336, r = 0.462, r = 0.437, r = 0.358, and r = 0.378, respectively, P < 0.05), and also between RT and TTP and CD31 vascular area (r = 0.415, and r = 0.421, respectively, P < 0.05). VEGFR1 and VEGFR2 expression did not correlate with any of the TIC parameters. CONCLUSIONS: CE-EUS with TIC analysis enables minimally invasive assessment of CRC angiogenesis and may provide information regarding the lymph nodes invasion. However, further studies are needed for defining its role in the evaluation of CRC patients.

Correlations between endothelial cell markers CD31, CD34 and CD105 in colorectal carcinoma.

PURPOSE: Colorectal carcinoma is an important cause of mortality worldwide. The fact that tumor growth is dependent on angiogenesis has supported researches for new prognostic parameters and the development of novel therapeutic strategies. Accordingly, we sought to evaluate angiogenesis quantitatively by assessing microvessel density in colorectal cancer. MATERIALS AND METHODS: The blood vessels stained with CD31, CD34 and CD105 were counted, and we reported their number per square millimeter in order to obtain microvascular density (MVD). Then, we aimed at comparing the performance of three endothelial cell markers (CD31, CD34, and CD105) on formalin-fixed tissues from 58 patients diagnosed with colorectal cancer. RESULTS: Following the comparison of the average effective vessels marked with the three markers, Student's t-test showed that the mean number of blood vessels marked with CD34 is higher than the blood vessels marked with CD31 and CD105. A significant difference that has been registered between the three levels of the T stage was found in the patients in our study, in terms of value marker CD105, ANOVA p=0.049, which returns to a value <0.05. Quick time decreases the pT stage, the observed differences being close to statistical significance. However, the result of ANOVA test does not allow us to say that differences can be generalized and not just a particular result, valid only for the study group, p=0.061 >0.05. There is a significant difference between patients with stage T, in terms of value: hemoglobin (ANOVA p<0.001), hematocrit (ANOVA p<0.001), mean corpuscular volume (MCV) (ANOVA p<0.001), mean corpuscular hemoglobin (MCH) (ANOVA p=0.002 <0.01 - significant difference with 99% confidence). By calculating the Pearson's correlation coefficient for the relationship CD31-CD105, we obtained a value r=0.440, which corresponds to p=0.0013 <0.05, indicating a statistically noteworthy direct correlation between the two factors. CONCLUSIONS: CD31 marker increases simultaneously with the CD105, in the cases analyzed throughout the present study. The ability of tumors to maintain a high vascular blood density in their inner portions may represent a reliable parameter to evaluate tumor angiogenesis and a finding relevant for future development of therapeutic angiogenesis strategies.

The influence of irradiation on autophagy process in normal and malignant colorectal epithelia.

AIM: The authors assessed the influence of preoperative radiotherapy on autophagy process using a quantitative assessment of LC3 expression on both normal and tumoral colorectal tissues. MATERIALS AND METHODS: Normal and malignant tissue samples were taken from 50 patients that underwent surgery for colorectal adenocarcinoma of which 11 received preoperative radiotherapy. Tissue samples were included in paraffin and sections were immunomarked for LC3 expression. LC3 percentage was assessed with dedicated software on 10 randomly selected fields with 40× objective from both normal and malignant tissue samples of each patient. The resulting data were assessed and compared with a statistical apparatus. RESULTS: LC3 was overexpressed in tumoral tissue as compared with normal one. The LC3 percentage is different from person to person and the higher it is in normal epithelium, the higher will be in tumoral epithelium of the same person, regardless the irradiation. The LC3 expression levels are decreasing from tumoral non-irradiated epithelia to normal irradiated epithelia. LC3 expression in tumoral cells is granular, with particular perinuclear disposal and often "annular" pattern. CONCLUSIONS: The autophagy process has a basal level in the normal tissue, with interindividual variability. The autophagy process proved to be upregulated in the tumoral cells, with a particular morphological expression, namely the presence of cytoplasmic coarse granules disposed in an "annular" pattern. Preoperative radiotherapy is downregulating the autophagy process both in normal and tumoral tissue but to a lesser extent in the latter.

Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease.

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients.

Preliminary study of correlations between the intratumoral microvessel density and the morphological profile of colorectal carcinoma.

AIM: New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. The aim was to evaluate intratumoral vascular density (ITMVD) and to analyze possible correlations between ITMVD and the morphological profile of colorectal carcinoma. MATERIALS AND METHODS: The studied group consisted of 50 patients that underwent surgery for colorectal tumors, 12 of them receiving preoperatory radiotherapy. The analyzed morphological parameters were tumor site, tumor gross aspect, tumor longitudinal and transverse diameter, tumor grading, local invasion (pT), regional invasion (pN), distant metastases (pM) and intratumoral microvessel density (ITMVD) expressed as number of capillaries÷mm². The malignant tissue samples were included in paraffin blocks and serial tissue sections were cut both for Hematoxylin-Eosin staining and CD34 immunomarking. For each case, five consecutive fields without necrosis were randomly selected with ×10 objective. Quantitative measurements were performed using special software for image analysis. RESULTS: For non-irradiated colorectal tumors, ITMVD was the highest in rectal localization, in infiltrative tumors, in circumferential tumors, in tumors with low longitudinal extension, in moderately differentiated (G2) tumors and in pT4, pN0 and pM1 tumors. DISCUSSION: Correlations showed different trends of ITMVD depending on each parameter: ITMVD was higher when the tumor was closer to the rectum, when it was more infiltrative, more circumferential or with low longitudinal diameter. These trends might be exploited in defining future anti-angiogenic therapeutic strategies. CONCLUSIONS: There were some interesting correlations between ITMVD and studied morphological parameters that have to be validated on larger series of cases.

Determination of autophagy gene ATG16L1 polymorphism in human colorectal cancer.

Autophagy has emerged not only as an essential repair mechanism to degrade damaged organelles and proteins but also as a major player in protection of tumor cells from multiple stresses. It was shown that autophagy gene polymorphisms are correlated with development of chronic inflammatory lesions, which represent a risk factor for colorectal tumors. In this study, we aimed to determine if ATG16L1 +898A>G (Thr300Ala) polymorphism is associated with an increased risk of developing colorectal cancer (CRC) and to establish correlations between ATG16L1 genotypes and the major clinical and morphological parameters. We observed that subjects carrying GG genotype were at a higher risk for CRC (OR 1.99, 95% CI: 1.02-3.91, p=0.039) when compared with the more frequent AA genotype, furthermore this was even more consistent in male subjects (OR 2.72, 95% CI: 1.11-6.63, p=0.019) but not in female subjects (OR 1.29, 95% CI: 0.43-3.86, p=0.652). In addition, we noticed a correlation between ATG16L1 GG genotype and tumor stage in moderately and poorly differentiated CRC cases. GG genotype carrying patients were at a higher risk for CRC (OR 5.19, 95% CI: 1.50-17.87, p=0.002) when compared with the more frequent AA genotype. Such correlation suggests a possible role of autophagy gene polymorphisms in the development of human colorectal cancer.

Analysis of aortic size in subjects died due to cardiovascular and non-cardiovascular events: a necropsy study.

Aortic size is known to be a predictor for cardiovascular deaths. The purpose of this study was to investigate whether aortic diameters measured on tissue samples obtained during autopsy from subjects that died of cardiovascular disease were bigger when compared to those from subjects deceased from other cause than cardiovascular disease. The study included 91 deceased subjects (average age 56 ± 18.1 years), which underwent autopsy to determine cause of death. Morphological measurements were completed on 364 aortic specimens obtained from four different sites, namely ascending aorta, aortic arch, distal thoracic aorta and abdominal aorta. Aorta showed the tendency to decrease in diameter from ascending aorta to abdominal aorta, the latter presenting with the smallest diameter. All studied aortic diameters were found to be larger in the cardiovascular population (p<0.01).