DNA chip technology in brain banks: confronting a degrading world

DNA microarray technology is based on the principle of hybridization between 2 complementary strands of nucleic acids, one being fixed into a solid membrane, the other being the sample to analyze. This has resulted in a very powerful method to examine differential gene expression between samples, and has been widely used in the study of tumors. The application of DNA microarray technology to the study of the nervous system has to consider several properties of the nervous tissue: composition of various neuronal types, as well as astrocytes, oligodendrocytes, and microglia; regional and area differences; developmental and age-dependent variations; and functional and pathological status. Moreover, human samples are usually obtained postmortem following variable agonal periods and postmortem delays between death and tissue preservation, which are accompanied by variable RNA degradation. Yet human postmortem nervous tissue stored in brain banks offers a unique opportunity to facilitate material for the study of diseases of the nervous system and to gain direct understanding on the mechanisms of disease. This review analyzes the application of DNA microarray technology to current practice using brain-banked tissues in order to recognize and minimize sub-optimal processing of brain samples and to correct pitfalls due to inadequate procedures. Also discussed are RNA preservation and RNA degradation effects on expression pattern assessments, analysis of individual versus pooled samples, array normalization, types of DNA chip platforms, whole genomic analysis versus specialized chips, and microgenomics. Minimizing RNA degradation and improving detection of resistant RNA in postmortem brain has been considered in detail in order to improve the efficiency and reliability of DNA microarray technology employed in the study of human postmortem nervous tissue.     

Short-term brain atrophy changes in relapsing-remitting multiple sclerosis

The objective of this study was to establish whether the time interval of 3 months is sufficient to detect whole-brain atrophy changes in patients with relapsing-remitting (RR) multiple sclerosis (MS). Another aim was to assess the value of monthly gadolinium (Gd)-enhanced magnetic resonance imaging (MRI) and of different Gd-enhancement patterns as predictors of brain atrophy. Thirty patients with RRMS (mean disease duration 4.9 years, mean age 34.4 years and mean Expanded Disability Status Scale [EDSS] 1.4) were assessed at baseline and monthly for a period of 3 months with clinical and MRI examinations. Calculations of baseline and monthly absolute and percent changes of MRI measures have been obtained using two semiautomated (Buffalo and Trieste) and one automated (SPM99) segmentation method. Changes of brain parenchymal fraction (BPF) were investigated according to Gd-enhancement patterns. Mean absolute and percent changes of BPF did not significantly differ at any time point in the study for any of the three methods. There was slight but not significant decrease of BPF from baseline to month 3: -0.0004 (0.05%), p=0.093 for Trieste; -0.0006 (0.07%), p=0.078 for Buffalo; and -0.0006 (0.08%), p=0.081 for SPM99 method. In ring-enhancement positive patients, there was a significant difference between baseline and month 3 changes of BPF, EDSS, and number of relapses. Over the study period, we did not demonstrate differences between changes of BPF according to the presence of Gd enhancement. Longitudinally, multiple regression analysis demonstrated that the only clinical or MRI parameter that predicted BPF decrease was the mean absolute change of ring-enhancing lesion load (R=0.62, p=0.003). The noteworthy findings of this study are (1) the observation that a significant brain atrophy progression cannot be detected over a 3-month period in RRMS; (2) the demonstration that the ring-enhancement pattern may contribute to more severe brain tissue loss in the short term; and (3) the lack of relationship between the presence and duration of Gd-enhancement activity and brain volume changes in the short term.     

Corticosteroids treatment

Corticosteroids (Cs) are widely used for treatment of multiple sclerosis (MS) acute relapses because of the potent immunosuppressive and anti-inflammatory properties. As for patients with relapsing-remitting (RR) MS, short-term administrations of Cs markedly less severity of symptoms and promote faster recovery of clinical attacks. Chronic administrations of Cs significantly diminish the formation of T1 hypointense lesions and the progression of brain atrophy. As for patients with secondary progressive MS treatment with Cs delays the time to onset of sustained disability. Finally the association between methylprednisolone and interferon beta (IFNbeta) leads the recovery of active lesions at greater extent and reduces the formation of neutralizing antibodies (NABs) against IFNbeta in patients with RRMS.     

Time-course variations of oxyradical metabolism, DNA integrity and lysosomal stability in mussels, Mytilus galloprovincialis, during a field translocation experiment

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study, several biomarkers were investigated in marine mussels caged for 4 weeks into an industrialised harbour of north-west Italy. Organisms were collected at different time intervals to better characterise the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants (catalase, glutathione S-transferases, glutathione reductase, total glutathione), the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralise specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidised bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with no variation or increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment. These findings suggest an initial counteractive response of mussels toward the enhanced prooxidant challenge, while antioxidants appeared overwhelmed at longer exposure periods. The hypothesis of reactive oxygen species (ROS) mediated toxicity is supported by the appearance of cell damages (DNA integrity and lysosome membrane stability), which exhibited a progressive enhancement during the course of the experiment with a maximum impairment after 30 days of exposure.     

Direct medical costs unequivocally related to diabetes in Italian specialized centers

This study estimated the resource utilization and direct medical costs in Italian diabetes centers (DCs). Hospital admissions for major chronic complications were not considered since DCs deliver primary care and follow up only complications unequivocally related to diabetes-acute complications and diabetic foot. The multicenter, prospective, observational study involving 31 Italian DCs included a total of 1,910 patients classified into eight prognostic groups by type of diabetes (types 1 and 2), metabolic control (HbA1c >7.5%, HbA1c 7.5%) and age (60, >60). The average total cost of type 1 diabetes per patient per year ranged from € 762 in group 2 (age 60, HbA1c >7.5%) to € 1,060 in group 4 (age >60, HbA1c >7.5%), and that the cost of type 2 diabetes from € 423 in group 5 (age 60, HbA1c 7.5%) to € 613 in group 8 (age >60, HbA1c >7.5%). The study brought to light the wide variability in the single cost components across clinically defined groups of patients. The cost of diabetes management in the strict sense was significantly affected by the type of diabetes and metabolic control.Data monitoring: E. Negri.E. Ansaldi, Alessandria; C. Baggiore, Florence; M. Balsanelli, Ostia; C. Bertoni, La Spezia; V. Borzì, Catania; A. Boscolo Bariga, Chioggia; A. Bruno, Turin; S. Caronna, Parma; F. Chiaromonte, Rome; S. Ciaccio, Pisa; G. Cicioni, Terni; M. Di Mauro, Catania; S. Gamba, Turin; L. Gentile, Asti; S. Giannini, Florence; D. Giorgi Pierfranceschi, Piacenza; T. Lavagnini, Padua; M. Lunetta, Catania; M. Marchesi, Bolzano; I. Meloncelli, San Benedetto del Tronto (Ascoli Piceno); G. Micali, Messina; M. Orrasch, Treviso; C. Pacchioni, Modena; M. Parillo, Caserta; G. Perriello, Perugia; S. Pistone, Potenza; G. Rinaldi, Naples; G. Sessa, Naples; M. Tagliaferri, Larino; P. Tatti, Marino (Rome); P. Ubaldi, Genua; M. Velussi, Monfalcone (Triest); E. Vitacolonna, Pescara; G. Zoppini, Verona; P. Zucchi, Asola (Mantua).     

Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family

Genetic variation in the ABCA4 (ABCR) gene has been associated with several distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration. The current model of genotype/phenotype association suggests that patients harboring deleterious mutations in both ABCR alleles would develop RP-like retinal pathology. Here we describe ABCA4-associated phenotypes, including a proband with a homozygous nonsense mutation in a family from Southern Italy. The proband had been originally diagnosed with STGD. Ophthalmologic examination included kinetic perimetry, electrophysiological studies and fluorescein angiography. DNA of the affected individual and family members was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray. A homozygous nonsense mutation 2971G>T (G991X) was detected in a patient initially diagnosed with STGD based on funduscopic evidence, including bull's eye depigmentation of the fovea and flecks at the posterior pole extending to the mid-peripheral retina. Since this novel nucleotide substitution results in a truncated, nonfunctional, ABCA4 protein, the patient was examined in-depth for the severity of the disease phenotype. Indeed, subsequent electrophysiological studies determined severely reduced cone amplitude as compared to the rod amplitude, suggesting the diagnosis of CRD. ABCR400 microarray is an efficient tool for determining causal genetic variation, including new mutations. A homozygous protein-truncating mutation in ABCA4 can cause a phenotype ranging from STGD to CRD as diagnosed at an early stage of the disease. Only a combination of comprehensive genotype/phenotype correlation studies will determine the proper diagnosis and prognosis of ABCA4-associated pathology.     

Syphilitic interstitial keratitis: treatment with immunosuppressive drug combination therapy

OBJECTIVE: The following is a case presentation of congenital syphilitic keratitis in a boy 6 years of age who was successfully treated with an immunosuppressive drug combination therapy. METHODS: Congenital syphilitic keratitis was diagnosed by clinical findings and laboratory tests. The child was unresponsive to traditional treatment; thus, systemic immunosuppressive therapy, which consisted of oral cyclosporine 4 mg/kg/d, 6 days per week, and oral low-dose steroids (fluocortolone 0.8 mg/kg a week, given every other day), was initiated. RESULTS: Corneal disease showed great improvement with this therapy, with progressive healing of lesions in the first month of treatment and no signs of toxic renal, hepatic, or growth abnormalities. Recurrences of uveitis have not occurred, and corneal interstitial keratitis episodes have been limited to 3 in an 8-year period. After 6 months with no recurrences, a tapering off of the systemic therapy was initiated, and the child is still asymptomatic and without flare-ups. CONCLUSIONS: Congenital syphilitic keratitis is usually treated with topical steroids and cycloplegic drugs, which not only can be ineffective but can also lead to complications such as cataract and glaucoma. In the present case report, a pediatric patient affected by syphilitic interstitial keratitis was treated successfully with an immunosuppressive drug combination therapy.     

Nicotine exerts a permissive role on NMDA receptor function in hippocampal noradrenergic terminals

The coexistence of nicotinic cholinergic receptors (nAChRs) and of N-methyl-D-aspartate (NMDA) receptors on the same noradrenergic axon terminals and the nAChR/NMDA receptor cross-talk were investigated by monitoring the release of noradrenaline (NA) evoked in superfused rat hippocampal synaptosomes by (-)-nicotine and NMDA alone or in combination. In medium containing a physiological concentration (1.2 mM) of Mg2+, the release of [3H]NA was very slightly increased by NMDA plus glycine, whereas it was significantly enhanced by (-)-nicotine. The (-)-nicotine/NMDA combination elicited supraadditive release which was totally abolished by the nAChR blocker mecamylamine and partly prevented by selectively blocking NMDA receptors. Supraadditive [3H]NA release was also observed by exposing synaptosomes to veratrine, but not to ionomycin. The supraadditive release elicited by the (-)-nicotine/NMDA or the veratrine/NMDA combination was sensitive to the protein kinase A/C inhibitor staurosporine and the selective protein kinase A inhibitor H89, but insensitive to the protein kinase C inhibitor Ro 31-8220. It is concluded that (i) release-modulating nAChRs and NMDA receptors coexist on hippocampal noradrenergic axon terminals; and (ii) nicotine permits NMDA receptor activation in the presence of Mg2+, possibly because the nicotine-induced influx of Na+ depolarizes the nerve ending membrane sufficiently to remove the Mg2+ block.