MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

Responses to GHRH plus arginine test are more concordant with IGF-I circulating levels than responses to arginine and clonidine provocative tests.

BACKGROUND/OBJECTIVE: Although pharmacological GH stimulation tests are still considered the gold standard for GH deficiency (GHD) diagnosis, they are burdened by poor specificity. The majority of children diagnosed as having GHD show normal GH responses when re-tested at the end of growth, thus questioning the initial diagnosis. We evaluated the concordance between IGF-I levels and GH responses to provocative tests. METHODS: We analyzed 105 GHRH plus arginine tests, 79 arginine tests, and 124 clonidine tests performed in 192 short children. IGF-I levels ≤-2SD score (SDS) were considered suggestive for high likelihood of GHD. The percentage of positive and negative results for each test was determined and compared with IGF-I levels, clinical follow-up and response to therapy. RESULTS: In children with IGF-I>-2SDS the arginine test showed a concordance rate of 6.9%, the clonidine test of 28.6%, and GHRH plus arginine test of 70%. In children with IGF-I≤-2SDS the concordance was 96.1%, 85.7%, and 46.4%, respectively. The overall concordance was 66.7% for GHRH plus arginine, 42.7% for clonidine, and 27.8% for arginine tests. CONCLUSION: Our results suggest that GHRH plus arginine test provides the best concordance with the assessment of IGF-I levels thus suggesting that the combination of the two procedures may significantly reduce the need of a second provocative test.

The assessment of human health impact caused by industrial and civil activities in the Pace Valley of Messina.

The impact of industrial and civil activities on an agricultural and residential area is presented in a detailed and global analysis. The examined area is the Pace river valley situated in the northern zone of Messina (Italy). The sources of pollution present in the area are: a Municipal Solid Waste Incinerator operating since 1979, a disused urban solid waste landfill which was used for 30 years, an urban solid waste treatment facility with heavy vehicles traffic, and two open pits for the production of bitumen. Large quantities of toxic, carcinogenic substances and criteria pollutants are released into the environment and represent potential hazards to human health. The analysis is performed using the EHHRA-GIS tool which employs an integrated, multimedia, multi-exposure pathways and multi-receptor risk assessment model that is able to manage all the steps which constitute the human health risk analysis in a georeferenced structure. The transport of pollutants in different environmental media is assessed applying models (AERMOD, GMS, CALINE) that take into account the particular three-dimensional morphology of the terrain. The results obtained, combined with a probabilistic risk assessment and a sensitivity analysis of calculation parameters, are a comprehensive assessment of the total human health risk in the area. Finally human health risks caused by toxic and carcinogenic substances are compared with acceptable legal limits in order to support environmental managers' decisions.

Catch-up growth in body mass index is associated neither with reduced insulin sensitivity nor with altered lipid profile in children born small for gestational age.

OBJECTIVE: Low birth weight is a risk factor for coronary heart disease. Persons who have coronary events as adults tend to have been small at birth and thin at 2 yr of age, after which they tended to increase their body mass index (BMI). Our aim was to determine whether BMI gain is associated to alterations in insulin sensitivity and/or lipid profile in children born small for gestational age (SGA). DESIGN: Retrospective case-control study. METHODS: We studied 78 children (mean age 7.8+/-2.5 yr): 26 SGA children with catch-up growth in BMI (CGB-SGA) (BMI= 10th to 75th centile), 26 SGA without catch-up growth (NCGB-SGA) (BMI<10th centile), and 26 appropriate for gestational age (AGA) control children (BMI: 10th to 75th centile). For each CGB-SGA child, we selected an NCGB-SGA and an AGA child of the same gender, age (within 1 yr), and pubertal status. SGA children were also subdivided into 2 groups according to post-natal catch-up growth in height (CGH). RESULTS: Glucose was significantly lower in NCGBSGA than AGA group (p=0.02). No significant differences in fasting insulin, fasting glucose/insulin ratio, homeostasis model assessment, quantitative insulin-sensitivity check index, and lipid profile were found among the 3 groups. HDL-cholesterol proved significantly reduced in SGA children with post-natal CGH (p=0.02). CONCLUSIONS: Our findings do not support the hypothesis of early alterations in insulin sensitivity and lipid metabolism in CGB-SGA subjects during childhood provided that BMI remains within the normal range. Finally, the finding of reduced HDL-cholesterol levels in CGH-SGA children suggests detrimental metabolic effects of the height gain.

Growth hormone therapy does not alter the insulin-like growth factor-I/insulin-like growth factor binding protein-3 molar ratio in growth hormone-deficient children.

BACKGROUND: Recent studies have linked raised levels of IGF-I and/or reduced levels of its main binding protein, IGF binding protein (IGFBP)-3, with the risk of developing cancer. A GH dose-dependent increase in IGF-I/IGFBP-3 molar ratio has been reported in subjects treated with GH, raising concern about the long-term safety. OBJECTIVE: The aim of this study was to evaluate changes in serum IGF-I, IGFBP-3, and IGF-I/IGFBP-3 molar ratio over the first 12 months of replacement GH therapy in GH deficient (GHD) children. METHODS: The study included 20 GHD children who had not previously received GH treatment, and 40 untreated non-GHD short children closely matched for age, gender, pubertal stage, and body mass index (BMI), as controls. Serum IGF-I, IGFBP-3 levels were measured before and after 12 months of GH treatment. Based on the molecular weight of IGF-I (7500) and IGFBP- 3 (40,000, mean of glycosylated variants), we calculated the molar ratio of IGF-I/IGFBP-3. RESULTS: IGF-I/IGFBP-3 molar ratio significantly increased during GH therapy (p=0.01). No significant difference in IGF-I/IGFBP-3 ratio was found between GHD children and controls at the different time points. In the multiple regression analysis, BMI (beta=0.33) and age (beta=0.33) proved to be the major predictors of the IGF-I/IGFBP-3 molar ratio (adjusted r2=0.53, p<0.0001). CONCLUSIONS: Our results suggest that at a conventional replacement dose GH does not alter the IGF-I/IGFBP-3 molar ratio. Potential fears related to long-term cancer risk are likely to be greatest in patients exposed to high-dose GH therapy and with genetic predisposition to high IGF-I and/or low IGFBP-3 concentrations.

Dual infections by influenza A/H3N2 and B viruses and by influenza A/H3N2 and A/H1N1 viruses during winter 2007, Corsica Island, France.

BACKGROUND: The investigation of dual influenza infection human cases is of major interest specifically for the control of new emerging influenza strains. OBJECTIVES: Using RT-PCR assays, we retrospectively assessed the prevalence of dual influenza virus infections that occurred in patients during the 2006-2007 winter season in Corsica Island (France). STUDY DESIGN: One hundred and thirty-four nasal swabbing samples taken from patients suffering from influenza-like illness between February and March 2007 were analysed using a rapid influenza antigen detection test, cell culture and RT-PCR assays. RESULTS AND CONCLUSION: Influenza viruses were detected in 93 (69.4%) of 134 patients with influenza-like illness using the combination of classical and molecular assays. Dual respiratory infections by influenza viruses were detected in 3 (3.2%) of the 93 influenza positive patients, including two cases of infection by influenza A/H3N2 and B viruses and one case of dual infection by influenza A/H3N2 and A/H1N1 viruses. In the present report, human co-infection cases by two influenza viruses appeared as a rare event in symptomatic patients. However, the virological and epidemiological mechanisms that determine the occurrence of dual influenza infections remain to be fully investigated in further prospective multicentric studies.

Measurement properties of the WOMAC LK 3.1 pain scale.

OBJECTIVE: The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) is applied extensively to patients with osteoarthritis of the hip or knee. Previous work has challenged the validity of its physical function scale however an extensive evaluation of its pain scale has not been reported. Our purpose was to estimate internal consistency, factorial validity, test-retest reliability, and the standard error of measurement (SEM) of the WOMAC LK 3.1 pain scale. METHOD: Four hundred and seventy-four patients with osteoarthritis of the hip or knee awaiting arthroplasty were administered the WOMAC. Estimates of internal consistency (coefficient alpha), factorial validity (confirmatory factor analysis), and the SEM based on internal consistency (SEM(IC)) were obtained. Test-retest reliability [Type 2,1 intraclass correlation coefficients (ICC)] and a corresponding SEM(TRT) were estimated on a subsample of 36 patients. RESULTS: Our estimates were: internal consistency alpha=0.84; SEM(IC)=1.48; Type 2,1 ICC=0.77; SEM(TRT)=1.69. Confirmatory factor analysis failed to support a single factor structure of the pain scale with uncorrelated error terms. Two comparable models provided excellent fit: (1) a model with correlated error terms between the walking and stairs items, and between night and sit items (chi2=0.18, P=0.98); (2) a two factor model with walking and stairs items loading on one factor, night and sit items loading on a second factor, and the standing item loading on both factors (chi2=0.18, P=0.98). CONCLUSION: Our examination of the factorial structure of the WOMAC pain scale failed to support a single factor and internal consistency analysis yielded a coefficient less than optimal for individual patient use. An alternate strategy to summing the five-item responses when considering individual patient application would be to interpret item responses separately or to sum only those items which display homogeneity.

The analysis of human health risk with a detailed procedure operating in a GIS environment.

An approach for quantifying the human health risk caused by industrial sources, which, daily or accidentally, emit dangerous pollutants able to impact on different environmental media, is introduced. The approach is performed by the HHRA-GIS tool which employs an integrated, multimedia, multi-exposure pathways and multi-receptors risk assessment model able to manage all the steps of the analysis in a georeferenced structure. Upper-bound excess lifetime cancer risk and noncarcinogenic hazards are the risk measures, the spatial distribution of which is calculated and mapped on the involved territory, once all the pathways and receptors of the study area are identified. A sensitivity analysis completes the calculations allowing to understand how risk estimates are dependent on variability in the factors contributing to risk. The last part of the paper makes use of a case study concerning a working industrial site to put in evidence in which way the designed tool can help local authorities and policy makers in managing risks and planning remedial and reduction actions. The considered geographical area is a hypothetical territory characterized by residential, agricultural and industrial zones. The presence of two sources of contamination, a municipal waste incinerator (MWI) and a contaminated site, are evaluated by the tool application. Various typologies of receptors have been taken into account, each of them characterized by different anatomical and dietary properties. The achieved results are analyzed, compared with acceptable and background values and alternatives of minor environmental impact calculated.

Urinary excretion of pyridinium crosslinks in short children treated with growth hormone.

The aim of this study was to evaluate the effect of growth hormone (GH) treatment on bone resorption in children with GH deficiency and those with idiopathic short stature. The study population included seven children with subnormal spontaneous GH secretion and 13 children with idiopathic short stature, all of them pre-pubertal. Anthropometric measurements, free, protein-bound and total urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd), serum GH, and serum immunoreactive PTH were measured at baseline and months 1, 3, 6 and 12 of GH treatment. The urinary excretion of total Pyd and Dpd, standardized by the cube of height (m3) in overnight, 24-hour urine collections was not different from age-matched healthy controls at baseline in either group of patients. During treatment with human recombinant GH, both pyridinium crosslinks increased above normal values, reaching a peak after one month in children with GH deficiency and later (after 3-6 months) in children with short stature. Free and total crosslink forms were correlated, and GH treatment did not affect the proportion of free to bound crosslinks. Serum concentrations of iPTH showed a moderate but not statistically significant increase. This study provides no evidence of reduced bone resorption in untreated GH deficiency or in idiopathic short stature. GH treatment induced a marked, but temporary, increase of bone resorption in both groups of patients.

2-N-acylaminoalkylindoles: design and quantitative structure-activity relationship studies leading to MT2-selective melatonin antagonists.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands.

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

Long-term treatment with growth hormone improves final height in a patient with Pallister-Hall syndrome.

Pallister-Hall syndrome is a disorder of development consisting of hypothalamic hamartoma, pituitary dysfunction, central polydactyly and visceral malformations. This disorder is inherited as an autosomal dominant trait and is caused by mutations of the GLI3 gene encoding a zinc finger transcription factor. We describe a case of Pallister-Hall syndrome with growth hormone neurosecretory dysfunction, successfully treated with growth hormone until attainment of final height. We conclude that children with Pallister-Hall syndrome and short stature be evaluated carefully for spontaneous somatotropic function and, if necessary, treated with growth hormone.

A new melatonin receptor ligand with mt1-agonist and MT2-antagonist properties.

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt1 and MT2. In the present work, we report the synthesis and pharmacological characterization of a new compound N-¿2-[5-(2-hydroxyethoxy)-1H-indol-3-yl)] ethyl¿ acetamide or 5-hydroxyethoxy-N-acetyltryptamine (5-HEAT). To assess the activity of the compound, the following tests were performed: affinity determination for the high- and low-affinity receptor states (2-[125I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([35S]GTPgammaS binding assay). 5-HEAT showed little selectivity for the mt1 receptor, with pKi values of 7.77 for mt1 and 7.12 for the MT2 receptors, respectively. 5-HEAT was able to differentiate between the high- and the low-affinity receptor states in the mt1 but not in the MT2 receptor. 5-HEAT induced a high level of G protein activation when acting through the mt1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT2 receptor-mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin-induced MT2 receptor-mediated G protein activation, with a pKB value of 7.4. In conclusion, it appears that 5-HEAT possesses very different efficacies at the two melatonin receptors, behaving as a full melatonin receptor agonist at the mt1 and as an antagonist/weak partial agonist at the MT2 receptor. Therefore, it is a promising ligand for use in functional studies aimed at distinguishing between the effects mediated by the different melatonin receptors in the human.

Design and synthesis of melatonin receptors agonists and antagonists.

We review our work towards the design and synthesis of high-affinity melatonin (N-acetyl-5-methoxytryptamine) agonist and antagonist compounds. High affinity melatonergic agonists were obtained by shifting the melatonin side chain from C3 to N1 of the indole ring system. Conversely, by moving the side chain from C3 to C2 it was possible to obtain melatonin antagonist compounds, albeit of moderate affinity.

Hazardous materials transportation: a risk-analysis-based routing methodology.

This paper introduces a new methodology based on risk analysis for the selection of the best route for the transport of a hazardous substance. In order to perform this optimisation, the network is considered as a graph composed by nodes and arcs; each arc is assigned a cost per unit vehicle travelling on it and a vehicle capacity. After short discussion about risk measures suitable for linear risk sources, the arc capacities are introduced by comparison between the societal and individual risk measures of each arc with hazardous materials transportation risk criteria; then arc costs are defined in order to take into account both transportation out-of-pocket expenses and risk-related costs. The optimisation problem can thus be formulated as a 'minimum cost flow problem', which consists of determining for a specific hazardous substance the cheapest flow distribution, honouring the arc capacities, from the origin nodes to the destination nodes. The main features of the optimisation procedure, implemented on the computer code OPTIPATH, are presented. Test results about shipments of ammonia are discussed and finally further research developments are proposed.

Through ARIPAR-GIS the quantified area risk analysis supports land-use planning activities.

The paper first summarises the main aspects of the ARIPAR methodology whose steps can be applied to quantify the impact on a territory of major accident risks due to processing, storing and transporting dangerous substances. Then the capabilities of the new decision support tool ARIPAR-GIS, implementing the mentioned procedure, are described, together with its main features and types of results. These are clearly shown through a short description of the updated ARIPAR study (reference year 1994), in which the impact of changes due to industrial and transportation dynamics on the Ravenna territory in Italy were evaluated. The brief explanation of how results have been used by local administrations offers the opportunity to discuss about advantages of the quantitative area risk analysis tool in supporting activities of risk management, risk control and land-use planning.

3-(2-Carbamoylvinyl)-4,5-dimethylpyrrole-2-carboxylic acids as ligands at the NMDA glycine-binding site: a study on the 2-carbamoylvinyl chain modification.

Twenty 4,5-dimethylpyrrole-2-carboxylic acids (5a-t) with different 2-carbamoylvinyl chains in position 3 were prepared to further investigate the relationships between structure and in vitro affinity for the strychnine-insensitive glycine-binding site. None of these compounds was superior to (E)-3-(N-phenyl-2-carbamoylvinyl)-4,5-dimethylpyrrole-2-carb oxylic acid III (pKi = 6.70), which was taken as a reference standard, but overall the results obtained indicate that the N-phenyl-2-carbamoylvinyl substituent of III may be replaced with the N-(1-adamantyl)-2-carbamoylvinyl group as in 5h (pKi = 6.20) without considerable loss of affinity. This finding adds to previous knowledge.

Melatonin.

Melatonin is the principal hormone secreted by the pineal gland, produced in humans with a circadian rhythm characterized by elevated blood levels during the night. It is involved in the regulation of several rhythmic functions in various vertebrates, and participates in the processing of photoperiodic information. Although its role in human physiologic and pathologic processes is not yet completely understood, MLT exerts a number of actions, in physiological or pharmacological concentrations, which could be of interest for future therapeutic uses. The mechanisms involved in MLT actions include interaction with membrane receptors, recently classified as mt1/MT2/MT3, and with nuclear sites corresponding to orphan members of the nuclear receptor superfamily, RZR/ROR; MLT also acts as a radical scavenger, exerting a protective action against various oxidative injuries. The present review is mainly addressed to the medicinal chemistry of ligands at the MLT membrane receptors, focusing on the models of binding interaction published in the literature. Several different pharmacophore and 3D-QSAR models have been reported so far, and a re-consideration of known active compounds, in the light of the recently developed biological tests on cloned receptors, could help to resolve the incongruities among these models; to this end, additional information is becoming available from new, conformationally constrained ligands, and from antagonist compounds with a selective affinity for receptor subtypes.

Structure-affinity relationships of indole-based melatonin analogs.

This paper reviews our progress made in characterizing structure-affinity relationships of indole-based melatonin analogs. Evidence is presented suggesting a preferred folded conformation for the amido side chain, almost orthogonal to the plane of indole. A 3D-QSAR comparative molecular field analysis (CoMFA) model, accounting for the observed differences in binding affinity within different classes of melatonergic ligands, and capable of quantitatively predicting the binding affinity of new compounds, is also reported.

Indole melatonin agonists and antagonists derived by shifting the melatonin side chain from the C-3 to the N-1 or to the C-2 indole position.

This article reviews our efforts in the development of indole melatonin (MLT) agonist and antagonist compounds. Evidence is presented which indicates that high-affinity melatonergic agonists were obtained by shifting the MLT amido side chain from the C-3 to the N-1 indole position. Conversely, by moving the side chain from the C-3 to the C-2 indole position it is possible to produce MLT antagonist compounds.