Genomic aberrations in normal appearing mucosa fields distal from oral potentially malignant lesions

Objectives

Oral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the ??field carcinogenesis?? model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy.

Methods

Nineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs).

Results

The frequencies of DNA aneuploidy (DI????1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI????1 and DI????1.4), while for d-OPMLs were high-aneuploid (DI?>?1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31?Cq13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31?Cq13.33 were associated with DNA aneuploidy (p?=?0.00004; p?=?0.0005; p?=?0.01).

Conclusions

ODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31?Cq13.33 may contribute to DNA aneuploidization.     

Headache at high school: clinical characteristics and impact

Although migraine (MH) and tension type headache (TTH) are the most common and important causes of recurrent headache in adolescents, they are poorly understood and not recognized by parents and teachers, delaying the first physician evaluation for correct diagnosis and management. The purpose of this study is to assess the knowledge about headache impact among the students of a Communication Private High School in Rimini city, and to evaluate the main different types of headaches interfering with school and social day activities. A self-administered questionnaire interview was given to students of the last 2 years of high school; ten items assessed the headache experience during the prior 12 months, especially during school time: the features and diagnosis of headaches types (based on the 2004 IHS criteria), precipitating factors, disability measured using the migraine disability assessment (MIDAS); therapeutic intervention. Out of the 60 students, 84 % experienced recurrent headache during the last 12 months. 79 % were females, aged 17-20 years; a family history was present in 74 % of headache students, in the maternal line; 45 % of subjects were identified as having MH and 27 % TTH; 25 % had morning headache and 20 % in the afternoon; fatigue, emotional stress and lack of sleep were the main trigger factors for headache, respectively in 86, 50 and 50 % of students; 92 % of headache students could not follow the lessons, could not participate in exercises and physical activity because of the headache; none had consulted a medical doctor and the 90 % of all students had never read, listened or watched television about headache. This study remarks on the need to promote headache educational programs, starting from high school, to increase communication between teachers-family-physician and patient-adolescents, with the goal to have an early appropriate therapeutic intervention, improvement of the quality of life and to prevent long-term headache disease in the adult age.     

Hyoscine-resistant peristalsis in guinea-pig ileum

The effect of hyoscine on the peristaltic activity of the proximal and distal ileum of the guinea-pig was studied. Hyoscine strongly impaired peristalsis as indicated by the elevation of the threshold pressure and by the increased number of incomplete peristalses and blockades. Functional activity of the circular musculature was more markedly impaired. However, particularly in the distal ileum, complete peristalses occurred even after 70 min exposure to hyoscine at a concentration of 10^?6 g/ml. A tenfold increase in hyoscine concentration failed to producedfurther impairment of peristaltic activity and of the oral reflex contraction. The activity which remained in the presence of hyoscine was blocked by methysergide and by d-tubocurarine. The hypothesis is advanced that once the muscarinic receptors have been blocked, increased radial stretch of the circular coat results in activation of a separate, tetrodotoxin sensitive, excitatory nervous pathway, which is sufficient to maintain a discrete degree of peristaltic activity.     

Human immunodeficiency virus type 1 (HIV-1) derived vectors: safety considerations and controversy over therapeutic applications

The latest generation of lentiviral vectors based on HIV-1 is one of the most efficient tools for gene transduction of mammalian cells. However, the possible employment of HIV-based vectors in clinical trials is a very controversial issue, mainly due to safety and ethical concerns. HIV-1 is a lethal pathogenic agent, which induces AIDS. Genetic vectors must derive either from viruses that are not pathogenic in humans, or that eventually just cause mild illnesses. Patients exposed to HIV-based vectors will test seropositive to certain components of HIV-1. In addition, there might be other possible adverse effects in patients that cannot be predicted, as many aspects of the pathogenesis of AIDS have not been completely understood yet. On these grounds, it seems necessary to improve the design of other lentiviral vectors, which derive from viruses that are not pathogenic in humans and are distantly related to primate retroviridae.     

Activation and internalization of the mu-opioid receptor by the newly discovered endogenous agonists, endomorphin-1 and endomorphin-2

The multiple effects of opiate alkaloids, important therapeutic drugs used for pain control, are mediated by the neuronal miro-opioid receptor. Among the side effects of these drugs is a profound impairment of gastrointestinal transit. Endomorphins are opioid peptides recently isolated from the nervous system, which have high affinity and selectivity for micro-opioid receptors. Since the miro-opioid receptor undergoes ligand-induced receptor endocytosis in an agonist-dependent manner, we compared the ability of endomorphin-1, endomorphin-2 and the micro-opioid receptor peptide agonist, [D-Ala2,MePhe4,Gly-ol5]-enkephalin (DAMGO), to induce receptor endocytosis in cells transfected with epitope-tagged micro-opioid receptor complementary DNA, and in myenteric neurons of the guinea-pig ileum, which naturally express this receptor. Immunohistochemistry with antibodies to the FLAG epitope or to the native receptor showed that the micro-opioid receptor was mainly located at the plasma membrane of unstimulated cells. Endomorphins and DAMGO induced micro-opioid receptor endocytosis into early endosomes, a process that was inhibited by naloxone. Quantification of surface receptors by flow cytometry indicated that endomorphins' and DAMGO stimulated endocytosis with similar time-course and potency. They inhibited with similar potency electrically induced cholinergic contractions in the longitudinal muscle-myenteric plexus preparation through an action antagonized by naloxone. The apparent affinity estimate of naloxone (pA2 approximately 8.4) is consistent with antagonism at the micro-opioid receptor in myenteric neurons. These results indicate that endomorphins directly activate the micro-opioid receptor in neurons, thus supporting the hypothesis that they are ligands mediating opioid actions in the nervous system. Endomorphin-induced micro-opioid receptor activation can be visualized by receptor endocytosis.     

Influence of fluoxetine and litoxetine on 5-HT4 receptor-mediated relaxation in the rat isolated oesophagus

The influence of two selective serotonin reuptake inhibitors (SSRIs), litoxetine and fluoxetine, has been studied on 5-HT4 receptor-mediated relaxation in the rat isolated oesophageal muscularis mucosae. In carbachol-precontracted oesophageal tissues, 5-hydroxytryptamine (5-HT) (0.1 nM-1 microM) induced concentration-dependent relaxations. Concentration-response curves were monophasic and reproducible. Litoxetine at concentrations without antimuscarinic properties (10 nM-1 microM) caused concentration-dependent relaxations, which reduced carbachol tone up to 37%. Higher litoxetine concentrations (3 microM-300 microM) were associated with marked relaxation up to the abolition of carbachol tone. The overall curve profile of litoxetine was biphasic in nature with a high (10 nM-1 microM) and a low (3 microM-300 microM) potency phase. Unlike 5-HT, the second curve of litoxetine was not reproducible, with a reduction involving mainly the low potency phase. Compared to litoxetine, fluoxetine caused minimal relaxation (less than 10% at 1 microM). Treatment of rats with parachlorophenylalanine (pCPA: 375 mg kg-1 per day, for two days), to deplete endogenous 5-HT stores, did not modify the relaxant effect of 5-HT, while it significantly reduced the high potency phase of litoxetine. In tissues from untreated rats, this phase was reduced by the 5-HT4 receptor antagonist GR 125487 (10 nM) to an extent similar (P = 0.20: ANOVA for continuous-by-class effects) to that induced by pCPA treatment. However, in tissues from pCPA treated animals GR 125487 (10 nM) exerted a slight but significant antagonism of litoxetine response (P = 0.037: ANOVA for continuous-by-class effects) mainly involving the high potency phase. In tissues from untreated rats, litoxetine (1 microM) increased the relaxant effects of 5-HT, while in tissues from pCPA treated animals it exerted a small but significant depression of the maximal response to 5-HT, without changing its potency value. Fluoxetine (1 microM) slightly, but significantly, antagonized the relaxant effect of 5-HT in an unsurmountable manner. In conclusion, litoxetine up to 1 microM relaxed the rat isolated oesophageal muscularis mucosae through a mechanism involving release of endogenous 5-HT, which in turn activates 5-HT4 receptors. However, based on results with GR 125487 in tissues from pCPA treated rats, a small component of litoxetine-induced relaxation may involve a direct activation of 5-HT4 receptors. It is unlikely that blockade of 5-HT reuptake can participate in the action of litoxetine, since fluoxetine, a 5-HT reuptake inhibitor equipotent to litoxetine, was ineffective in the same range of concentrations. The antimuscarinic activity of litoxetine, previously demonstrated in the isolated guinea-pig intestine, played a role at concentrations greater than 1 microM. The 5-HT-releasing action of litoxetine could account for the potentation by litoxetine of 5-HT-induced relaxation in tissues from untreated rats, which was reversed by pCPA treatment. Under these conditions, litoxetine depressed relaxations to high 5-HT concentrations only. In tissues from untreated rats, fluoxetine slightly but unsurmountably antagonized 5-HT-induced relaxations, thus confirming previous observations in the guinea-pig small intestine.     

Thymidylate synthase expression in normal colonic mucosa: a predictive marker of toxicity in colorectal cancer patients receiving 5-fluorouracil-based adjuvant chemotherapy

OBJECTIVES: We retrospectively evaluated the relevance of thymidylate synthase (TS) expression in normal colonic mucosa as a predictive factor of toxicity in colorectal cancer patients receiving adjuvant fluorouracil (5-FU)-based chemotherapy. METHODS: TS expression was immunohistochemically assessed on normal colonic mucosa from 50 patients with colorectal cancer Dukes' stages B (15 patients) and C (35 patients) treated with 5-FU-based adjuvant chemotherapy. RESULTS: The incidence of grade 2-3 diarrhea and stomatitis (according to WHO) was demonstrated to be significantly higher in patients with low nuclear TS expression in normal mucosa than in those with high TS expression (p < 0.0001). Moreover, patients with low TS expression in normal colonic mucosa developed weight loss and worsening of the performance status (according to ECOG score) more commonly than patients with high TS expression (p = 0.005 and p = 0.02, respectively). Furthermore, low TS expression in normal colonic mucosa significantly correlated with a higher rate of a delay of chemotherapy courses, dose reduction and treatment discontinuation. CONCLUSIONS: Immunohistochemical TS expression in normal colonic mucosa may represent an important predictive parameter for identifying a subset of patients with a high risk of developing severe 5-FU-related toxicities.     

Management of puberty in constitutional delay of growth and puberty

Constitutional delay of growth and puberty (CDGP) is the most common presenting form of short stature, but no single test can infallibly discriminate CDGP and isolated hypogonadotrophic hypogonadism. Management of puberty in CDGP aims to optimise not only growth maintaining body proportions and improving peak bone mass without impairing growth potential--but also well-being; for example, the distress boys often suffer because of their lack of growth and pubertal progression can affect their school performance and social relationships. Typical sex steroid treatments to induce puberty in boys with CDGP include testosterone (T) enanthate, T undecanoate, mixed T esters, T transdermal patches, and oxandrolone p.o. Compared with other regimens, short-course low-dose depot T i.m. is an effective, practical, safe, well tolerated, and inexpensive regimen. Some unresolved problems in management include optimal timing and dose of sex steroid treatment, the role of GH in CDGP, and the management of CDGP in girls.     

Refining the therapeutic range of posaconazole and isavuconazole for efficient therapeutic drug monitoring using a bioassay approach

Therapeutic drug monitoring (TDM) of antifungal triazole was recommended, except for isavuconazole (ISA) whose target trough concentrations (C_min) need to be specified. Concerning posaconazole (POS), tablet formulation results in higher exposure but no upper C_min threshold has been yet proposed. We aimed to investigate the pharmacokinetic–pharmacodynamic relationship of POS and ISA, using a bioassay approach as surrogate marker of antifungal activity, in order to refine the therapeutic C_min of both antifungals. A bioassay using a cellulose disk diffusion method was performed to determine the growth inhibition zone (GIZ) of POS and ISA on Aspergillus fumigatus and Candida parapsilosis (ISA only). GIZs of plasma from patients undergoing TDM for POS (n = 136) or ISA (n = 40) were determined. GIZs of plasma patients and antifungal C_min were highly correlated for ISA (A. fumigatus: ρ = 0.942, P < 0.0001; C. parapsilosis: ρ = 0.949, P < 0.0001) and POS (ρ = 0.922, P < 0.0001), and these relationships were represented with a Michaelis–Menten model. Based on this modeling, the recommended thresholds of 0.7, 1, and 1.25 mg/L for the POS C_min corresponded to 50.1, 55.2, and 59.1% of the maximal GIZ, respectively. We propose an upper threshold of 4.8 mg/L for the POS C_min and a lower threshold of 2.0 mg/L for the C_min of ISA, as they respectively corresponded to concentrations leading to 90% and 50% of the maximal GIZ on A. fumigatus. The determination of antifungal activity using this bioassay allowed refining target C_min of POS and ISA, especially the upper threshold of POS (4.8 mg/L) and the lower threshold of ISA (2.0 mg/L).