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The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

Objectives: To evaluate whether intrauterine growth restriction (IUGR) as seen in preeclampsia is associated with high levels of fetal DNA in maternal circulation, and whether fetal DNA is related to altered uterine and/or umbilical artery Doppler velocimetry., Methods: Fetal DNA quantification was performed by real-time PCR on SRY sequences in 64 male-bearing pregnant women with IUGR and/or preeclampsia and 89 controls., Results: Fetal DNA content was significantly elevated in IUGR pregnancies similar to preeclampsia and correlated with altered umbilical Doppler velocimetry, while no correlation was found with uterine Doppler status., Conclusion: Increased fetal DNA levels in maternal plasma may be a sign of placental or fetal pathology even in the presence of normal uterine Doppler velocimetry, allowing a more precise diagnostic evaluation. The finding that elevated fetal DNA in IUGR pregnancies correlates with abnormal umbilical Doppler velocimetry suggests that fetal DNA release is associated more with fetal chronic hypoxia than with fetal size.

The levels of the soluble (s) CD40:sCD40 ligand (L) dyad, which belongs to the tumor necrosis factor (TNF)-alpha:TNF-alpha-receptor superfamily, are significantly increased in the cerebrospinal fluid (CSF), but not the serum of cobalamin (Cbl)-deficient (Cbl-D) rats. They were normalized or significantly reduced after treatment with Cbl, transforming growth factor-beta1 or S-adenosyl-L-methionine, and the normal myelin ultrastructure of the spinal cord was concomitantly restored. The concomitance of the two beneficial effects of these treatments strongly suggests that the increases in CSF sCD40:sCD40L levels may participate in the pathogenesis of purely myelinolytic Cbl-D central neuropathy in the rat. In keeping with this, an anti-CD40 treatment prevented myelin lesions.

Background: Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous disorders characterized by progressive spasticity of the lower limbs. Mutations in the SPG4 gene, which encodes spastin protein, are responsible for up to 45% of autosomal dominant cases., Objective: To search for disease-causing mutations in a large series of Italian patients with HSP., Design: Samples of DNA were analyzed by direct sequencing of all exons in SPG4. Samples from a subset of patients were also analyzed by direct sequencing of all exons in SPG3A, SPG6, SPG10, and SPG13., Setting: Molecular testing facility in Italy., Patients: Sixty unrelated Italian patients with pure (n = 50) and complicated (n = 10) HSP., Main Outcome Measures: Mutations in SPG4, SPG3A, SPG6, SPG10, and SPG13., Results: We identified 12 different mutations, 8 of which were novel, in 13 patients. No mutations of any of the other HSP genes tested were found in 15 patients with sporadic pure HSP who did not have mutations in the SPG4 gene., Conclusions: The overall rate of mutation in the SPG4 gene within our sample was 22%, rising to 26% when only patients with pure HSP were considered. The negative result obtained in 15 patients without mutations in SPG4 in whom 4 other genes were analyzed (SPG3A, SPG6, SPG10, and SPG13) indicate that these genes are not frequently mutated in sporadic pure HSP.

We have recently demonstrated that the neuropathological morphological alterations caused by cobalamin (Cbl) deficiency in the rat central nervous system are related to the vitamin's inability to modulate the synthesis of some neurotoxic and neurotrophic agents in opposite directions. In the present study, we measured nerve growth factor (NGF) levels in the spinal cord (SC) and cerebrospinal fluid (CSF) of rats made Cbl-deficient (Cbl-D) by means of total gastrectomy (TG) or a Cbl-D diet. In both cases, Cbl deficiency increased SC and CSF NGF levels after the appearance of myelinolytic lesions in the SC white matter (SCWM) (i.e. after the second post-TG month), and these changes were normalised by Cbl treatment in the 4-month-totally-gastrectomised (TGX) rats. Intracerebroventricular (i.c.v.) anti-NGF-antibody treatment prevented the onset of the myelinolytic SCWM lesions in the 2-month-TGX rats (i.e. when SC and CSF NGF levels are still normal) and normalised the ultrastructure of the SCWM in the 4-month-TGX rats, which was however worsened by the i.c.v. administration of NGF. These findings demonstrate that: (i) Cbl deficiency increases SC and CSF NGF levels; and (ii) endogenous NGF seems to play a noxious role in the progression of rat Cbl-D central neuropathy.

Mitochondrial deoxyribonucleic acid depletion syndromes are autosomal recessive disorders characterized by a reduction of the amount of mitochondrial deoxyribonucleic acid, which impairs the synthesis of respiratory chain complexes. Mutations in the deoxyguanosine kinase and polymerase gamma genes have been identified in hepatocerebral forms, whereas thymidine kinase 2 gene mutations have been found in patients with isolated myopathy, encephalomyopathy, or spinal muscular atrophy. Mutations in the gene encoding the beta subunit of the adenosine diphosphate-forming succinyl-coenzyme A synthetase have also been reported in a family. In this report, the clinical, molecular, morphologic, and biochemical features of five children from two independent families with an infantile encephalomyopathy are characterized. The affected children manifested muscle mitochondrial deoxyribonucleic acid depletion and three novel thymidine kinase 2 gene mutations. They consist of a homozygous substitution resulting in Ala to Val change at the highly conserved position 181 of thymidine kinase in the first family, and two heterozygous substitutions in the second family: a Cys to Trp change at residue 108 and a Leu to Pro change at residue 257 of the enzyme. Common clinical features associated with these TK2 mutations are a normal early developmental phase followed by psychomotor regression, encephalopathy often with epileptic seizures, and myopathy with features of a progressive dystrophic process.

Objective: To define and differentiate psychological and adjustment problems due to brain injury or brain tumour in children and adolescents., Methods: Two groups of patients with acquired brain lesions (24 post-traumatic patients and 22 brain tumour survivors), ranging in age between 8-15 years, received a psychological evaluation, including the Child Behaviour Checklist for Ages 4-18 (CBCL) and the Vineland Behaviour Adaptive Scales (VABS)., Results: Both groups showed psychological and social adjustment problems. Post-traumatic patients were more impaired than brain tumour survivors. Social adjustment problems were associated to externalizing problems in post-traumatic patients and internalizing problems in brain tumour surviving patients., Conclusions: These differences in psychological and behavioural disorders between the two groups must necessarily be considered when developing psychological treatment, rehabilitation plan and social re-entry.

The distribution of the Glu298Asp polymorphism in NOS3 gene was determined in 405 Italian patients with "probable" Alzheimer's disease (AD) compared with 253 age-matched controls. Total plasma homocysteine (tHcy) levels were evaluated in 97 patients and 23 controls, and were correlated with the Glu298Asp genotype. A significantly increased frequency of the Glu/Glu genotype in late onset AD (LOAD) patients was found. tHcy levels were significantly increased in patients compared with controls and, notably, higher in LOAD than in early onset AD (EOAD). Stratifying by the Glu298Asp genotype, a trend toward an increase of tHcy was present in Glu/Glu homozygous. This wild type genotype seems to be associated with LOAD. tHcy levels are significantly increased in AD compared with controls and, moreover, higher in LOAD than in EOAD, possibly in correlation with the microvascular disease occurring with aging. Besides, a contribution of the Glu/Glu genotype in increasing tHcy levels has been observed.

Background: We tested the hypothesis that acetyl-L-carnitine (ALC) may have a protective and a curative role in chemotherapy-induced hyperalgesia in vivo, in animal models of cisplatin-, paclitaxel- and vincristine-induced neuropathy. In addition, the possible interaction between ALC and vincristine antineoplastic action was assessed., Materials and Methods: Chemotherapy-induced peripheral neuropathy (CIPN) was induced in different groups of rats. The effect of ALC was evaluated both when its administration was started together with the administration of anticancer drugs ("preventive" protocol) and when ALC administration was started later on during treatment ("curative" protocol)., Results: The ALC treatment significantly prevented the lowering of the mechanical nociceptive threshold when the administration started concomitantly and, respectively, with cisplatin, paclitaxel and vincristine as compared to each drug alone. Furthermore, when ALC administration was started later on during treatment, at well-established neuropathy, ALC was able to restore the mechanical nociceptive threshold within a few days. Finally, experiments indicated that ALC does not interfere with the antitumor effects of vincristine., Conclusion: Considering the absence of any satisfactory treatment currently available for CIPN in a clinical setting, these are important observations, opening up the possibility of using ALC to treat a wide range of patients who have undergone chemotherapy and developed sensory peripheral neuropathy.

Cognitive and psychological disorders are among the most frequently observed sequelae in brain tumor survivors. The goal of this work was to verify the presence of these disorders in a group of children and adolescents diagnosed with brain tumor before age 18 years, differentiate these disorders according to age of assessment, identify correlations between the two types of impairments and define possible associations between these impairments and clinical variables. The study involved 76 patients diagnosed with brain tumor before age 18 years. Three age groups were formed, and all the patients received a standardized battery of age-matched cognitive and psychological tests. According to our findings, all three groups present with cognitive and psychological-behavioral disorders. Their frequency varies according to age of onset and is strongly associated to time since diagnosis. The performance intelligence quotient (PIQ) was more impaired than the verbal intelligence quotient (VIQ). Internalizing problems, withdrawal and social problems were the most frequent psychological disorders. Correlations were found between cognitive impairment and the onset of the main psychological and behavioral disorders. These findings are relevant as they point out the long-term outcome of brain tumor survivors. Hence, the recommendation to diversify psychological interventions and rehabilitation plans according to the patients' age., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

Extremely variable clinic and genetic features characterize mitochondrial encephalomyopathy (MEM). Pathogenic mitochondrial DNA (mtDNA) defects can be divided into large-scale rearrangements and single point mutations. Clinical manifestations become evident when a threshold percentage of the total mtDNA is mutated. In some MEM, the "mutant load" in an affected tissue is directly related to the severity of the phenotype. However, the clinical phenotype is not simply a direct consequence of the relative abundance of mutated mtDNA. Other factors, such as nuclear background, can contribute to the disease process, resulting in a wide range of phenotypes caused by the same mutation. Using Affymetrix oligonucleotide cDNA microarrays (HG-U133A), we studied the gene expression profile of muscle tissue biopsies obtained from 12 MEM patients [4 common 4977 bp deleted mtDNA and 8 A3243G: 4 progressive external ophthalmoplegia (PEO) and 4 mitochondrial myopathy, encephalopathy, lactic cidosis, and stroke-like episodes syndrome (MELAS) phenotypes] compared with age-matched healthy individuals. We found several differentially expressed genes: 35 were markedly up-regulated in the mtDNA macro-deletion group (vs. the control group) and 4 decreased; 56 genes were dysregulated in A3243G-related disorders (53 down-regulated in PEO and 3 up-regulated in MELAS). Finally, 12 genes were similarly regulated in the majority of the MEM patients under study. Amongst these, we identified an increased expression of genes related to the metabolism of the amino groups, as well as of several genes involved in genetic information processing. Moreover, few genes were similarly decreased in MEM patients vs. the control group. Real-time PCR demonstrated excellent reproducibility of the microarray-based findings. The observed expression changes are likely to represent a molecular signature for mitochondrial disorders. Furthermore, the differential expression profile of MELAS(A3243G) vs. PEO(A3243G) may support a role of nuclear background in contributing to these different clinical phenotypes. MEM microarray data are available from GEO database (http://www.ncbi.nlm.nih.gov/geo/) with the accession number: GSE1462.

Converging evidence points to a pivotal role of vascular endothelial growth factor (VEGF) in neuronal protection and a lack of its activity in neurodegenerative disorders. To investigate this possible association, we screened the VEGF gene promoter for various well-known single-nucleotide polymorphisms in a series of 249 consecutively recruited Italian patients with sporadic Alzheimer's disease (AD). Genetic analysis indicated different distributions of two single-nucleotide polymorphisms in the AD population compared with healthy control subjects. In particular, the frequencies of -2578A/A and -1198C/T genotypes were significantly greater in AD patients than in control subjects (23.7 vs 14.7% and 2.8 vs 0%, respectively). The -2578A/A genotype was associated with an increased risk for disease, independently of apolipoprotein E genotype. The risk was significantly increased with respect to various VEGF genotype combinations. In contrast, no difference in serum VEGF levels was detected comparing 96 patients and 49 control subjects. These findings suggest that polymorphisms within the promoter region of the VEGF gene confer greater risk for AD, probably by reducing its neuroprotective effect, and confirm the biological role of VEGF in neurodegenerative processes.

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations., Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions., Results: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before., Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real-time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male-bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long-term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild-type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild-type allele signals.

We describe a new mitochondrial DNA mutation in a male infant who presented clinical and magnetic resonance imaging features of Leigh syndrome and died at the age of 9 mo. The patient's development was reportedly normal in the first months of life. At the age of 5 mo, he presented severe generalized hypotonia, nystagmus, and absent eye contact. Laboratory examination showed increased lactate and pyruvate in both serum and cerebrospinal fluid. Brain magnetic resonance imaging revealed multiple necrotic lesions in the basal ganglia, brain stem, and thalamus. Muscle histopathology was unremarkable, whereas respiratory chain enzyme analysis revealed a severe complex I deficiency. The patient died after an acidotic coma at age 9 mo. Sequence analysis of the entire mtDNA disclosed a new T10158C mutation with variable tissue heteroplasm (muscle: 83%; blood: 48%). The mutation was undetectable in the blood of his unaffected mother. The transition changes a serine residue into a proline, in a highly conserved region of the NADH dehydrogenase subunit 3 (ND3). This is the first description of a mitochondrial ND3 gene in Leigh syndrome with early lethality.

Mitochondrial encephalomyopathies are highly variable clinically and at the genetic level. In practice, when the mitochondrial DNA (mtDNA) of any mitochondrial-patient is sequenced, a very high number of variations are noted. The vast majority of these differences are simply polymorphisms, that is, non-pathologic, homoplasmic sequence variations; however, when a heteroplasmic variant is detected (co-existence of two different populations in the same tissue) this is clinically significant. We identified two different heteroplasmic mutations in the mtDNA of two subjects: G4298A in the tRNA(Ala) (Alanine) gene and T10010C in the tRNA(Gly) (Glycine), both of which have been reported previously. This work confirms the pathogenicity of these mutations and helps define the correlation between genotype and phenotype.

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis., (Copyright 2003 Wiley-Liss, Inc.)

Extra corporeal photochemotherapy (ECP) is an immunomodulating procedure used in several nonneurological diseases which, similarly to multiple sclerosis, are likely to be due to T-cell-mediated autoimmunity and it is probable that ECP can modulate the normal activity of peripheral blood mononuclear cells (PBMC). Using the Lewis rat experimental allergic encephalomyelitis (EAE) model of human multiple sclerosis (MS) we examined the effect of extracorporeal UV-A irradiation on psoralen-activated PBMC. In our experiment the comparison between the two groups of animals (ECP or sham-treatment) evidenced that the ECP treatment reduced the severity of EAE on clinical grounds and this result was confirmed by the pathological examination. The changes in the titers of anti-myelin antigen antibodies typical of EAE were also modulated by the procedure. Ex vivo examination evidenced a significant reduction in tumor-necrosis factor-alpha (TNF-alpha) released by PBMC after lipopolysaccharides (LPS) stimulation in culture. We conclude that ECP modifies the normal activity of PBMC during the course of EAE and it is possible that one of the anti-inflammatory mechanisms of action of ECP is correlated to a down-regulation of T-helper 1 lymphocytes activity.

Background: Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy., Objective: To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient., Methods: Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy., Results: CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia., Conclusions: Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.

Purpose: Antineoplastic drugs belonging to platinum or taxane families are severely neurotoxic, inducing the onset of disabling peripheral neuropathies with different clinical signs. Acetyl-L-carnitine (ALC) is a natural occurring compound with a neuroprotective activity in several experimental paradigms. In this study we have tested the hypothesis that ALC may have a protective role on cisplatin and paclitaxel-induced neuropathy., Experimental Design: Sensory nerve conduction velocity (SNCV) was measured in rats before, at end, and after an additional follow-up period from treatments with cisplatin, paclitaxel, or with the respective combination with ALC. In addition, serum from treated animals was collected to measure the levels of circulating NGF, and left sciatic nerves were processed for light and electron microscope observations. ALC interference on cisplatin and paclitaxel antitumor activity and protective mechanisms were investigated using several in vitro and in vivo models., Results: ALC cotreatment was able to significantly reduce the neurotoxicity of both cisplatin and paclitaxel in rat models, and this effect was correlated with a modulation of the plasma levels of NGF in the cisplatin-treated animals. Moreover, experiments in different tumor systems indicated the lack of interference of ALC in the antitumor effects of cisplatin and paclitaxel. The transcriptional profile of gene expression in PC12 cells indicated that ALC, in the presence of NGF, was able to positively modulate NGFI-A expression, a gene relevant in the rescue from tissue-specific toxicity. Finally, the transcriptionally ALC-mediated effects were correlated to increase histone acetylation., Conclusion: In conclusion, our results indicate that ALC is a specific protective agent for chemotherapy-induced neuropathy after cisplatin or paclitaxel treatment without showing any interference with the antitumor activity of the drugs.

Mitochondrial (mt)DNA alterations cause cellular energy failure and respiratory chain dysfunction. Single large-scale rearrangements represent the most common mtDNA mutations and are responsible for very variable clinical manifestations. Here, we show an increased frequency of the A12308G substitution, a common polymorphism used to define the European mtDNA haplogroup U, in mitochondrial patients carrying mtDNA single macrodeletion. In this group of patients, A12308G substitution is associated with a higher relative risk of developing pigmentary retinal degeneration, short stature, dysphasia-dysarthria and cardiac conduction defects. MtDNA haplotype might modulate the clinical expression of mitochondrial encephalomyopathies due to mtDNA macrodeletions.

Objective: To better understand the still unknown pathologic mechanism involved in the accumulation of multiple mtDNA deletions in stable tissues., Methods: A large-scale screening of mtDNA molecules from skeletal muscle was performed in 14 patients with progressive external ophthalmoplegia (PEO) and 2 patients with mitochondrial neurogastrointestinal encephalomyopathy carrying mutations on ANT1, C10ORF2 or POLG1, and TP genes., Results: Patients with at least one mutation in the exonuclease domain of POLG1 showed the highest frequency of individually rare point mutations only in the mtDNA control region; in addition, high levels, in terms of frequency and heteroplasmy, of recurrent mutations (A189G, T408A, and T414G) and alterations affecting the (HT)D310 region were detectable in many of the patients. Two homozygous POLG1 mutations, within the exonuclease domain, were able to induce an increased mutational burden also in fibroblasts from patients with PEO., Conclusions: Specific POLG1 mutations directly affect the integrity of the mtDNA by reducing its proof-reading exonuclease activity, resulting in the accumulation of heteroplasmic levels of both randomly rare and recurrent point mutations in the skeletal muscle tissue and fibroblasts.

The aim of the present study was to determine the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 in brainstem neurons following noxious visceral stimulation. Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid (ENS, UNS) or were left untreated (ECT, UCT). Paraffin embedded brain sections immunoreacted with an antibody specific for phosphorylated ERKs. In noxious stimulated rats ERKs activated neuron profiles in the periaqueductal gray matter, parabrachial, dorsal raphe, solitary tract nucleus, area postrema and superior colliculus suggest that ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The comparison between ENS and UNS rats shows that the long acting anaesthetic urethane attenuates the number of the ERKs activated neurons compared to the short acting ether.

A 13084 A->T missense mutation in the mitochondrial ND5 gene was identified in a 16-year-old boy affected with a progressive neurodegenerative disorder combining features of Leigh and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) syndromes. Muscle biopsy analysis revealed partial complex I deficiency. The mutation presented a variable degree of heteroplasmy in the patient's tissues. This finding underlines the contribution of mtDNA-encoded complex I subunits in the etiology of complex I deficiency associated with encephalopathy.

Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing.

We have identified a heteroplasmic G to A mutation at position 12,183 of the mitochondrial transfer RNA Histidine (tRNA(His)) gene in three related patients. These phenotypes varied according to mutation heteroplasmy: one had severe pigmentary retinopathy, neurosensorial deafness, testicular dysfunction, muscle hypotrophy, and ataxia; the other two had only retinal and inner ear involvement. The mutation is in a highly conserved region of the T(psi)C stem of the tRNA(His) gene and may alter secondary structure formation. This is the first described pathogenic, maternally inherited mutation of the mitochondrial tRNA(His) gene.

Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l-chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high-performance liquid chromatography (DHPLC) and double-gradient denaturing gradient gel electrophoresis (DG-DGGE). We analysed the sequence encoding the 5' untranslated flanking region of ferritin l-chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C-G transversions. DHPLC and DG-DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del-T156. We conclude that DHPLC, similar to DG-DGGE, detects all the mutations in the l-ferritin 5'UTR sequence in a single run, and that various mutations occur outside the IRE structure.

Objective: To quantitatively measure male DNA in blood from women with systemic sclerosis (SSc) and from controls and to evaluate in vitro the modulation of the microchimeric cell population size following immunologic stimuli that were expected to trigger antigen-specific T cells., Methods: A real-time polymerase chain reaction for a Y chromosome sequence was used to measure male DNA in blood from women with SSc and from controls who gave birth to sons. The in vitro change in the microchimeric cell population size was measured following immunologic stimuli, which were expected to trigger antigen-specific T cells., Results: Cellular microchimerism was found in SSc patients and controls, but the absolute amount of male DNA was higher in the patients, and the in vitro addition to blood mononuclear cells of an anti-CD28 costimulatory signal acted as a powerful amplification of microchimeric cells in 3 patients with SSc but not in controls., Conclusion: Cellular microchimerism is a physiologic phenomenon in parous women. In SSc patients, cellular microchimerism is accounted for by a higher number of cells that have the characteristics of T lymphocytes specific to maternal allogeneic antigens.

Mitochondria are involved in cellular energy production via oxidative phosphorylation and this function may be damaged by any mutation in mitochondrial DNA (mtDNA). To identify novel mtDNA mutations, we have developed a program to systematically screen the entire mitochondrial genome in a large number of individuals with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. The sequence-data were obtained with an automated rapid system, which gave us a series of information: in the eleven mitochondrial genomes analyzed we observed the presence of 33 differences from the revised Cambridge Reference Sequence (Andrews et al., 1999), but they were all homoplasmic in the patients' tissues analyzed (skeletal muscle and blood), suggesting that they are unlikely to be primarily pathogenic though they may be co-responsible in the determination of the disease. This work can therefore help complete the already ample mtDNA polymorphism existent database., (Copyright 2002 Wiley-Liss, Inc.)

Background: Oxaliplatin neurotoxicity represents a clinically-relevant problem and its etio-pathogenesis is still unknown. We explored the possible role of some neuronal growth factors ("neurotrophins") during the course of oxaliplatin sensory neuronopathy., Materials and Methods: In our rat model two different doses of oxaliplatin were used (2 and 3 mg/kg i.v. twice weekly for 9 times). The neurotoxicity of the treatment was assessed with neurophysiological and pathological methods and serum neurotrophin levels were measured by ELISA., Results: Both oxaliplatin-treated groups showed the neurophysiological and neuropathological changes which mimic the chronic effects of oxaliplatin administration in humans, e.g. reversible sensory impairment due to dorsal root ganglia neuron damage. These changes were associated with a significant and dose-dependent reduction only in the circulating level of nerve growth factor (NGF), which returned to normal values after neurophysiological and pathological recovery., Conclusion: This specific association between neurological impairment and NGF modulation indicates that NGF impairment has a role in the neurotoxicity of oxaliplatin.

We aimed at verifying whether extracellular signal-regulated kinases (erks) 1 and 2 are activated, i.e. phosphorylated, in forebrain neurons after visceral pain stimulation (VPS). Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid or were left untreated (ECT, UCT). After 2 h the animals were perfused. Paraffin embedded brain sections immunoreacted with an antibody selective for the phosphorylated erks. The light microscope analysis revealed only a few labelled neurons in ECT, while in UCT, positive cells were detected. In VPS rats (VPSR) positive cells were mainly distributed in regions, such as the hypothalamic anterior and thalamic paraventricular midline nuclei, amygdala, hippocampal and parahippocampal, insular and perirhinal cortex, involved in nociception and/or visceral activities. Our data suggest an association of erks activation with the emotional component of nociception; moreover, they show that erks activation is not suppressed by anaesthesia.

A severe muscle enolase deficiency, with 5% of residual activity, was detected in a 47-year-old man affected with exercise intolerance and myalgias. No rise of serum lactate was observed with the ischemic forearm exercise. Ultrastructural analysis showed focal sarcoplasmic accumulation of glycogen beta particles. The enzyme enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the beta-enolase subunit, the protein product of the ENO3 gene. The beta-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while alpha-enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues; a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant beta-enolase. Muscle beta-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

Two dual-task experiments are reported bearing on the issue of slower processing time for severe chronic closed-head injury (CHI) patients compared to matched controls. In the first experiment, a classical psychological refractory period (PRP) paradigm was employed, in which two sequential stimuli, a pure tone and a colored dot, were presented at variable stimulus onset asynchronies (SOAs), each associated with a distinct task. The task on the tone required a speeded vocal response based on pitch, and the task on the colored dot required a speeded manual response based on color. In the second experiment, either one or three masked letters was presented, followed by a pure tone at variable SOAs. The task on the letters required a delayed report of the letters at the end of each trial. The task on the tone required an immediate manual response based on pitch. In both experiments, both CHI patients and matched controls reported an SOA-locked slowing of the speeded response to the second stimulus, a PRP effect. The PRP effect was more substantial for CHI patients than for matched controls, suggesting that a component of the slower processing time for CHI patients was related to a selective increase in temporal demands for central processing of the stimuli.

It has been demonstrated that tyrosine kinase (TK) and phosphatidylinositol 3-kinase (PI3-K) are involved in IgE-mediated stimulation of human basophils; conversely, little is known about the biochemical pathways activated by IL-3 and GM-CSF. The aim of this study was to evaluate the effects of TK and PI3-K inhibitors on basophil histamine release induced by anti-IgE, IL-3 and GM-CSF. Since IL-3 and GM-CSF cause histamine release from normal human basophils only when the inhibitory effect of extracellular Na(+) has been removed, peripheral blood leukocytes were suspended in isotonic solutions containing either 140 mM NaCl or 140 mM N-methyl-D-glucamine(+). After stimulation with anti-IgE, IL-3 or GM-CSF, histamine release was measured by an automated fluorometric method. The effects of preincubation with four different TK inhibitors (AG-126, genistein, lavendustin A, tyrphostin 51) and one PI3-K inhibitor (wortmannin) were evaluated. AG-126, genistein and lavendustin A exerted a significant dose-dependent inhibitory effect on basophil histamine release induced by anti-IgE (either in high or in low Na(+) medium), IL-3 and GM-CSF. Among the TK inhibitors, lavendustin A exerted the most potent activity, followed by AG-126 and genistein. Tyrphostin 51 caused a weak inhibition of histamine release induced by IL-3, GM-CSF and anti-IgE in a low Na(+) medium, but not in a physiological Na(+)-containing medium. The PI3-K inhibitor wortmannin exerted the most effective inhibitory activity on the histamine release induced by the three agonists. The combined effects of lavendustin A and wortmannin were less than additive, suggesting that TK and PI3-K are involved in the same activation pathway in human basophils. These results suggest a possible role of TK and PI3-K in basophil histamine release induced by anti-IgE, IL-3 and GM-CSF. TK and PI3-K are indeed potential therapeutic targets for antiallergic drugs.

Background: The development of squamous cell carcinoma of the lower lip is an interesting model of photocarcinogenesis because of the structural and topographic characteristics of the lips. The purpose of this study was to evaluate the expression of immunohistochemical markers on the lips of patients with lower lip squamous cell carcinoma (LLSCC), compared with a control population., Methods: Of the 98 subjects involved in the study, 58 were suffering from squamous cell carcinoma of the lower lip. The remaining 40 acted as a control. The case studies were taken from six university and hospital dermatology and plastic surgery departments. Questionnaires were administered to assess the risk factors for LLSCC. The cases involving squamous cell carcinoma underwent surgical excision and punch biopsy specimens were obtained from 20 control patients. Tissues were prepared in 5-microm-thick sections to carry out the following immunohistochemical study: PCNA, p53, AgNOR, cyclin-D1, bcl-2., Results: The lower lip was the predominant location of squamous cell carcinoma, with the following factors playing important roles: chronic sun exposure, history of smoking, alcohol use and familial risk of cutaneous tumors. The male/female ratio in our survey was 5:1. The p53 protein was positive in approximately 50% of SCC cases and in 20% of controls. This protein is mostly associated with chronically photoexposed skin areas. AgNOR positivity increased with the loss of cellular differentiation; a progressive increase in size and a poorly defined shape were evident in poorly differentiated carcinomas., Conclusions: The results of this multicenter study showed that there is a noticeable difference in the expression of PCNA, p53, cyclin-D1, and AgNOR in tissues from patients with LLSCC and controls.

Idiopathic inflammatory myopathies (IIM) are muscle diseases of autoimmune pathogenesis characterized by mononuclear cell infiltration within muscle tissue. Since immune cell homing and accumulation at the site of antigenic challenge is usually mediated by chemokines, we evaluated the expression of 2 beta-chemokines--monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha)--by immunohistochemistry and polymerase chain reaction in muscles of polymyositis, inclusion body myositis, and dermatomyositis patients, and related their expression to immunopathological alterations in muscle. MCP-1 and MIP-1alpha transcripts were detected by PCR in all IIM muscles, but not in controls. By immunohistochemistry, the chemokines were found in all IIM muscle sections located in infiltrating inflammatory cells and also in neighboring extracellular matrix. The extent to which extracellular matrix was filled by each chemokine differed in each disease. In view of the known ability of chemokines to bind extracellular matrix and their possible synthesis by extracellular matrix components, we suggest that chemokine storage in the extracellular matrix can act as a microenvironmental factor amplifying lymphocyte activation and migration, thereby maintaining the autoimmune attack against unknown muscle antigens.

Background: Many patients with classic congenital muscular dystrophy have been found to have partial or total deficiency of the alpha2 chain of laminin 2 (merosin). This deficiency has mostly been studied using only 1 antibody against a fragment of the protein., Objectives: To characterize the expression of laminin alpha2 in the skeletal muscle of patients with laminin alpha2 deficiency using antibodies against 2 different portions of the protein and to correlate the immunochemical findings with clinical phenotype., Methods: We studied 4 patients with total lack of laminin alpha2 and 12 with partial laminin alpha2 deficiency with immunohistochemical techniques and Western blot analysis. We used antibodies recognizing an 80-kd fragment toward the C-terminus and a 300-kd fragment toward the amino-terminal. Patient characteristics examined were functional compromise, magnetic resonance imaging or computed tomography of the brain, electromyography, evoked potentials, and creatine kinase levels., Results: In 4 patients, immunohistochemical analysis revealed no reactivity to either antibody; in 2 patients, the 300-kd fragment alone was partially expressed; in 2 patients, the 80-kd fragment alone was partially expressed; and in 8 patients, both fragments were partially expressed. Immunoblot analysis revealed bands of reduced intensity and normal molecular weight generally corresponding to the immunohistochemical findings. Absence of both fragments or of one with reduction of the other always produced a severe clinical phenotype, while a milder clinical phenotype was observed when both fragments were partially expressed., Conclusions: Extent of laminin alpha2 deficiency in most cases correlates with clinical phenotype but not with peripheral and central white matter abnormalities. Skin biopsy specimens may reveal laminin alpha2 deficiency in patients who have normal laminin alpha2 levels in muscle biopsy specimens.

Canine X-linked muscular dystrophy (CXMD) is genetically homologous to Duchenne muscular dystrophy and shares the severe myopathy and lethal clinical development of the human disease. We used immunohistochemistry to characterize the time course of postnatal expression of adult fast, adult slow and developmental myosin in the muscle of CXMD dogs, carriers and healthy controls. We also characterized the expression of utrophin and dystrophin. This detailed immunolocalization study confirmed that postnatal muscle maturation is delayed in normal dogs compared to other animals and humans, and is only achieved at around 60 days. In CXMD dogs major derangement of myosin expression became evident from about 15 days; there was a selective loss of fibers expressing fast myosin and persistence of developmental fibers compared to controls. In carriers, the proportion of dystrophin-deficient fibers, which mainly expressed fast myosin, decreased with age. In controls and carriers utrophin was absent from muscle fiber surfaces in 2-day-old animals but present between 15 and 30 days, to mostly disappear by 60 days. In dystrophic animals, sarcolemmal expression of utrophin was more marked and persistent. That immature neonatal muscle from control dogs normally contains sarcolemmal utrophin may have implications for the success of utrophin up-regulation therapy to correct the dystrophic phenotype. The data of this study provide important baseline information for further studies on the development and progression of pathological changes in the muscle of CXMD dogs.

We evaluated transforming growth factor-beta1 (TGF-beta1) expression in the muscle of four laminin alpha2-negative, four laminin alpha2-positive and seven partial laminin alpha2-deficient congenital muscular dystrophy (CMD) patients, and compared it to Duchenne muscular dystrophy (DMD) patients and controls. TGF-beta1 mRNA levels in skeletal muscle from laminin alpha2-negative and laminin alpha2-positive CMD patients were significantly greater than in controls (P < 0.05 and P < 0.005, respectively), while in partial laminin alpha2-deficient muscular dystrophy patients the amount was not significantly higher than in controls (P > 0.1). The TGF-beta1 values were lower than those found in DMD, although the extent of fibrosis was greater in CMD than in DMD and controls. Our findings suggest that TGF-beta1 is involved in CMD muscle fibrosis, but differently from what we observed in DMD muscles as it seems not to be the major player in connective tissue proliferation.

Alcohol is noxious to the brain and peripheral nervous system. However, wine contains substances that may have positive biological and pharmacological effects. Resveratrol is the most studied and probably the most active of these substances. This naturally occurring compound, which is present in wine and grapes, reduces oxidative stress in neuronal-like cell cultures. We have shown that resveratrol induces phosphorylation of the mitogen-activated protein (MAP) kinase family members, extracellular regulated kinase 1 (ERK1) and ERK2, in the human neuroblastoma SH-SY5Y cells in vitro at much lower concentrations than those found in the plasma of rats after oral wine administration. MAP kinases are involved in numerous different aspects of signal transduction in the cells. In particular, phosphorylation of ERK2 has been related to the synaptic changes at the basis of memory and learning processes. These findings, together with our own, on resveratrol-induced activation of MAP kinases in human neuronal-like cells, and previously published epidemiological studies which have demonstrated an inverse relationship between moderate wine intake and dementia, suggest that wine (not alcohol) may have a positive effect on nervous cells.