Development of an in vitro model to study tooth cystogenesis.

AIM: To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development. METHODOLOGY: To form spheroid structures, various numbers (1 × 104 , 5 × 104 or 1 × 105 ) of epithelial cells (HaCaT and Cal27) were seeded in 96-well plates previously coated with 1.5% low-melting agarose. After 24 h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24-well plates previously coated with polymerized collagen and kept for up to 21 days. Images of spheroids were captured at each time-point (1, 5, 9, 15 and 21 days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One-way Anova was used for statistical analysis. RESULTS: Both epithelial cell lines were able to generate regular and circular spheroids after 24 h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P < 0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst. CONCLUSIONS: This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance.

Responsiveness of clinical outcome measures in Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease (CMT) is a very slowly progressive neuropathy which makes it difficult to detect disease progression over time and to assess intervention efficacy. Experience from completed clinical trials with ascorbic acid and natural history studies confirm difficulties in detecting such changes. Consequently, sensitive-to-change outcome measures (OMs) are urgently needed. METHODS: The relative responsiveness of clinical scales of the Italian-UK ascorbic acid trial (placebo arm) were assessed by using the standardized response mean (SRM), which is the ratio of the paired scores mean change over time to the standard deviation of the score change (0 is worst responsiveness). RESULTS: Little worsening of OM scores was found over 2 years. In detail, the primary OM of the trial, the CMT Neuropathy Score version 1 (CMTNSv1), showed low responsiveness (SRM 0.13). Some CMTNS items showed slightly greater responsiveness (CMT Examination Score 0.17; CMTNS Signs 0.19). Myometric assessments of handgrip and foot dorsiflexion strength were the most responsive (SRM -0.31 and -0.38, respectively). Amongst the other measures, the nine-hole peg test, which assesses upper limb functioning, showed the best sensitivity to change (SRM 0.28). CONCLUSIONS: Overall these OMs showed low or negligible responsiveness, confirming the need to improve current OMs and to develop novel ones for prognostic and interventional studies. However, handgrip and foot dorsiflexion myometry are worth retaining for future trials as they were the most responsive and are likely to be clinically relevant for patients.

Is overwork weakness relevant in Charcot-Marie-Tooth disease?

BACKGROUND: In overwork weakness (OW), muscles are increasingly weakened by exercise, work or daily activities. Although it is a well-established phenomenon in several neuromuscular disorders, it is debated whether it occurs in Charcot-Marie-Tooth disease (CMT). Dominant limb muscles undergo a heavier overload than non-dominant and therefore if OW occurs we would expect them to become weaker. Four previous studies, comparing dominant and non-dominant hand strength in CMT series employing manual testing or myometry, gave contradictory results. Moreover, none of them examined the behaviour of lower limb muscles. METHODS: We tested the OW hypothesis in 271 CMT1A adult patients by comparing bilateral intrinsic hand and leg muscle strength with manual testing as well as manual dexterity. RESULTS: We found no significant difference between sides for the strength of first dorsal interosseous, abductor pollicis brevis, anterior tibialis and triceps surae. Dominant side muscles did not become weaker than non-dominant with increasing age and disease severity (assessed with the CMT Neuropathy Score); in fact, the dominant triceps surae was slightly stronger than the non-dominant with increasing age and disease severity. DISCUSSION: Our data does not support the OW hypothesis and the consequent harmful effect of exercise in patients with CMT1A. Physical activity should be encouraged, and rehabilitation remains the most effective treatment for CMT patients.

The effect of minor constituents of olive oil on cardiovascular disease: new findings.

There has been much interest regarding the components that contribute to the beneficial health effects of the Mediterranean diet. Recent findings suggest that polyphenolic compounds found in olive oil are endowed with several biologic activities that may contribute to the lower incidence of coronary heart disease in the Mediterranean area.

Enzymatic assay for the determination of olive oil polyphenol content: assay conditions and validation of the method.

A new spectrophotometric assay for the determination of the polyphenolic content of olive oil is presented. It is a substrate-recycling assay for phenolic compounds that employs tyrosinase in the presence of excess NADH. The reaction of various phenols with the enzyme produces an o-quinone, which is detected by recycling between reactions with the enzyme and NADH. The method offers some advantages over the classical methods employed to determine the polyphenolic content of olive oil, that is, ease and reproducibility of the analysis, highly increased sensitivity, and selectivity toward phenolic compounds. The amount of total polyphenols was determined in virgin olive oils both with the Folin-Ciocalteu reagent and with the proposed enzymatic method. The results suggest a better estimation of the polyphenol content, as compared with the colorimetric method. This has to be attributed to the different reactivities of the two methods toward phenols and catechols. Finally, the enzymatic method demonstrates that there is a linear relationship between the olive oil phenolic content and the antioxidative capacity of oil extracts.

In vitro cytotoxicity to human cells in culture of some phenolics from olive oil.

The neutral red in vitro cytotoxicity assay was used to evaluate the comparative responses of human cells isolated from tissues of the oral cavity to olive oil phenolics. The cell lines used included normal gingival fibroblasts, immortalized, nontumorigenic gingival epithelial cells, and carcinoma cells from the salivary gland. No differences in the relative sensitivities to the phenolics amongst the three cell types were noted. In general, for all cell types, the sequence of increasing cytotoxicity was: oleuropein aglycone>oleuropein glycoside, caffeic acid>o-coumaric acid>cinnamic acid>>tyrosol, syringic acid, protocatechuic acid, vanillic acid. Cytotoxicity was noted only at phenolic concentrations far exceeding those attainable after habitual consumption, thus indicating that consumption of phenol-rich olive oil is safe.

Vitamin C and Charcot-Marie-Tooth 1A: Pharmacokinetic considerations.

Charcot-Marie-Tooth 1A disease (CMT1A) is a disease for which no drug treatments are available. In 2004, it was reported that ascorbic acid reduced the severity of neuropathy in transgenic mice overexpressing PMP22, an animal model of human CMT1A, compared with untreated mice. Based on those results, clinical trials were undertaken at different centers worldwide and four of them have been completed, but none of them resulted in significant improvements. Based on the pharmacokinetics of ascorbic acid, we propose that the randomized clinical trial carried out thus far confirmed the tight control of ascorbic acid's absorption and proved its tolerability at one and two years. The pharmacokinetic considerations discussed in this article might largely explain the disappointing results of the recent CMT1A trials.

Exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study.

OBJECTIVE: To assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol. METHODS: (i) Group without lesions. Smears obtained from the lower lip, border of the tongue and floor of the mouth of 31 control individuals (group I), 49 tobacco users (group II) and 27 tobacco/alcohol users (group III) were stained using the Papanicolaou method. The first 100 cells counted on each smear determined the maturation pattern and the keratinization index (KI). Analysis of variance (ANOVA) and the Tukey multiple comparison test were used for statistical analysis, at a 5% significance level. (ii) Group with lesions. Cytopathological and histopathological studies were conducted for 15 patients: eight with leucoplakia without epithelial dysplasia, two with epithelial dysplasia and five with squamous cell carcinoma. RESULTS: (i) Group without lesions. Statistical analysis revealed a smaller number of superficial cells with nuclei in all sites of the group of tobacco/alcohol users (group III) when compared to the control group (group I), and this difference was statistically significant (P<0.005). (ii) Group with lesions. The severity of histopathological findings increased with the increase in the number of cells of the deeper epithelial layers, with a statistically significant difference in the number of intermediate (P=0.013) and parabasal cells (P=0.049), which increased with the severity of the epithelial maturation disorder: leucoplakias with dysplasia had a greater number of intermediate and parabasal cells than leucoplakias without dysplasia; and the number in squamous cell carcinomas was greater than in leucoplakias with dysplasia. CONCLUSION: The maturation pattern of cells in the three anatomic sites showed changes that may be associated with the synergistic effect of tobacco and alcohol. Also, the severity of histopathological findings was associated with the increase in the number of cells in the deeper epithelial layers.

Free radical-scavenging properties of olive oil polyphenols.

Plants in the Mediterranean basin, such as vine and olive trees, have developed an array of antioxidant defences to protect themselves from environmental stress. Accordingly, the incidence of coronary heart disease and certain cancers is lower in the Mediterranean area, where olive oil is the dietary fat of choice. As opposed to other vegetable oils, extra virgin olive oil, which is obtained by physical pressure from a whole fruit, is rich in phenolic components that are responsible for the particular stability of the oil. We have investigated the scavenging actions of some olive oil phenolics, namely hydroxytyrosol and oleuropein, with respect to superoxide anion generation, neutrophils respiratory burst, and hypochlorous acid. The low EC50S indicate that both compounds are potent scavengers of superoxide radicals and inhibitors of neutrophils respiratory burst: whenever demonstrated in vivo, these properties may partially explain the observed lower incidence of CHD and cancer associated with the Mediterranean diet.

The kinetics of copper-induced LDL oxidation depend upon its lipid composition and antioxidant content.

Copper promotes oxidation of human low-density lipoprotein (LDL) through molecular mechanisms that are still under investigation. We employed native human LDL, phospholipid-containing delipidated LDL ghosts, or trilinolein-reconstituted, phospholipid-containing LDL to investigate both LDL oxidation and the associated process of copper reduction. Both LDL ghosts and trilinolein-reconstituted LDL were devoid of antioxidants and were extremely susceptible to AAPH-induced oxidation but, paradoxically, were rather resistant to copper-mediated oxidation. The dynamic reduction of Cu(II) to Cu(I) was quantitatively decreased in LDL ghosts and in trilinolein-reconstituted LDL, also lacking the initial rapid reduction and the subsequent inhibition phases, due to the absence of endogenous antioxidants. Conversely, the rate of copper reduction was linear and likely due to lipid peroxides, either already present or formed during copper-induced oxidation. We suggest that copper undergoes redox transitions in LDL by utilizing reducing equivalents originating from endogenous antioxidants and/or from lipid peroxides in the LDL lipid core.