Claudia Nesti

Role of Neck Ultrasonography in the Follow-Up of Children Operated on for Thyroid Papillary Cancer

The aim of this study was to evaluate the role of neck ultrasonography compared to (131)I whole-body scan (WBS) and circulating thyroglobulin (Tg) measurement after thyroid hormone withdrawal in the follow-up of children with thyroid papillary cancer, who had previously undergone total thyroidectomy for the diagnosis of neck lymph node metastases (LNM). Forty-five children were examined. Neck ultrasonography and diagnostic WBS were conclusive about the presence or absence of LNM in 35 patients. Diagnostic WBS revealed the presence of LNM in 6 cases not detected by neck ultrasonography; neck ultrasonography was positive in 3 cases that were negative at diagnostic WBS but confirmed by post-(131)I therapy WBS. One patient with suspicious neck lymphnodes at neck ultrasonography not confirmed by WBS was considered as a false-positive result of neck ultrasonography. Neck ultrasonography and thyroglobulin (Tg) were conclusive about the presence or absence of LNM in 29 patients. Tg was elevated in 10 subjects with negative neck ultrasonography (7 had also lung and/or mediastinic LNM). Tg was undetectable in 5 patients in whom the presence of LNM was confirmed by neck ultrasonography and WBS. In conclusion, our study in children demonstrates that neck ultrasonography can detect LNM that are not suspected by palpation, diagnostic WBS, or serum Tg determination. Furthermore, neck ultrasonography can pinpoint the anatomic site of the LNM.

Investigation of interactions between poly-l-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation

Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription – polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

Micronuclei assay and FISH analysis in human lymphocytes treated with six metal salts.

The capability of some metal compounds for inducing micronuclei (MN) in human lymphocytes was studied. In this investigation, Al (III), Cd (II), Hg (II), Sb (V), Te (VI), and Tl (I) salts were considered. The FISH (fluorescence in situ hybridization) technique with a centromeric probe was coupled with the MN assay in binucleated cells in order to detect both centromere‐positive MN (C+ MN) due to malsegregation phenomena and centromere‐negative MN (C− MN) due to chromosome breakage. The blood of two young nonsmoking male donors was employed for all experiments. In both donors, all the tested metal compounds, with the exception of Tl2SO4, showed a statistically significant increase of MN compared to controls, at least at one dose. FISH analysis revealed an increase in the fraction of C+ MN for Al, Cd, and Hg compounds, and of C− MN for the Sb salt; however, this was not a statistically significant increase. A different efficiency was observed for the different metal compounds, in particular, KSbO3 and CH3HgCl, which were highly genotoxic, whereas the others showed minimal effects. Environ. Mol. Mutagen. 34:279–284, 1999.

Oxidative stress biomarkers in mitochondrial myopathies, basally and after cysteine donor supplementation

Mitochondrial diseases are due to impairment of the mitochondrial respiratory chain. A plausible pathogenic mechanism leading to cellular dysfunction and phenotypic expression is oxidative stress, but there are surprisingly few clinical studies on this subject. Glutathione (GSH) deficiency has been reported in mitochondrial diseases, and the biosynthesis of glutathione depends on cysteine availability. We have examined oxidative stress biomarkers [advanced oxidation protein products (AOPP) and ferric reducing antioxidant power (FRAP)] in blood samples from 27 patients and 42 controls. AOPP levels were greater in patients than in controls (P value <0.00001). Therefore, we performed a double-blind cross-over study to evaluate if 30-day supplementation with a whey-based cysteine donor could modify these markers, reduce lactate concentration during aerobic exercise, or enhance muscular strength and quality of life. Treatment did not modify lactate concentration, clinical scale (MRC) or quality of life (SF-36), but significantly reduced oxidative stress levels. Our findings reinforce the notions that in mitochondrial diseases oxidative stress is important and can be reduced by administration of a cysteine donor. Oxidative stress biomarkers may be useful to detect redox imbalance in mitochondrial diseases and to provide non-invasive tools to monitor disease status.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

Morpho-functional characterization of human mesenchymal stem cells from umbilical cord blood for potential uses in regenerative medicine

Mesenchymal stem cells (MSCs) represent a promising source of progenitor cells having the potential to repair and to regenerate diseased or damaged skeletal tissues. Bone marrow (BM) has been the first source reported to contain MSCs. However, BM-derived cells are not always acceptable, due to the highly invasive drawing and the decline in MSC number and differentiative capability with increasing age. Human umbilical cord blood (UCB), obtainable by donation with a noninvasive method, has been introduced as an alternative source of MSCs. Here human UCB-derived MSCs isolation and morpho-functional characterization are reported. Human UCB-derived mononuclear cells, obtained by negative immunoselection, exhibited either an osteoclast-like or a mesenchymal-like phenotype. However, we were able to obtain homogeneous populations of MSCs that displayed a fibroblast-like morphology, expressed mesenchym-related antigens and showed differentiative capacities along osteoblastic and early chondroblastic lineages. Furthermore, this study is one among a few papers investigating human UCB-derived MSC growth and differentiation on three-dimensional scaffolds focusing on their potential applications in regenerative medicine and tissue engineering. UCB-derived MSCs were proved to grow on biodegradable microfiber meshes; additionally, they were able to differentiate toward mature osteoblasts when cultured inside human plasma clots, suggesting their potential application in orthopedic surgery.

Human dental pulp stem cells protect mouse dopaminergic neurons against MPP+ or rotenone

Parkinsons disease (PD) is a neurodegenerative disorder characterized by the progressive death of substantia nigra dopaminergic neurons that results in a regional loss of striatal dopamine (DA) levels. Dental pulp contains ex vivo-expandable cells called dental pulp stem cells (DPSCs), with the capacity to differentiate into multiple cell lineages. More interestingly, due to their embryonic origin, DPSCs express neurotrophic factors such as brain-derived neurotrophic factor, nerve growth factor and glial cell-derived neurotrophic factor. The aim of the present study was to investigate the neuroprotective effects of DPSCs against MPP+ (2.5, 5, and 10 μM) and rotenone (0.25, 0.5 and 1 μM) in an in vitro model of PD, using an indirect co-culture system with mesencephalic cell cultures. When mesencephalic cultures were challenged with MPP+ or rotenone, in the presence of DPSCs a statistically significant protective effect was observed at all the tested doses in terms of DA uptake. DPSCs protective effect on DA neurons was also confirmed by immunocytochemistry: an increased number of spared tyrosine hydroxylase (TH)+ cells was observed in co-culture conditions compared to controls, and neurons showed longer processes in comparison with mesencephalic cells grown without DPSCs. In conclusion, the co-culture with DPSCs significantly attenuated MPP+ or rotenone-induced toxicity in primary cultures of mesencephalic neurons. Considering that the direct contact between the two cell types was prevented, it can be speculated that neuroprotection could be due to soluble factors such as BDNF and NGF, released by DPSCs. Blocking BDNF and NGF with neutralizing antibodies, the neuroprotecting effect of DPSCs was completely abolished. Therefore DPSCs can be viewed as possible candidates for studies on cell-based therapy in neurodegenerative disorders.

Lack of association between mtDNA haplogroups and Alzheimer’s disease in Tuscany

Mitochondrial DNA (mtDNA) haplogroup-specific polymorphisms were previously related to several neurodegenerative diseases, including Alzheimer’s disease (AD). However, the precise role of mtDNA haplogroups in the neurodegenerative cascade leading to AD is still unclear. In this work we have genotyped predefined European mtDNA haplogroups in 209 patients with AD and 191 matched controls. In order to minimise the risk of “genetic contamination”, which could lead to false associations between gene markers and disease, we were careful to enrol in the study only patients and controls of clear Tuscan origin (with at least three generations of Tuscanborn relatives). The frequency of the haplogroups did not differ between the two groups, and no correlation with gender, ApoE genotype, age of onset or disease status was observed. Further studies will be required to define the contribution of mtDNA haplogroups, if any, to the pathogenesis of AD. A correct population selection, in order to minimise the risk of genetic contamination, is essential in these studies.

Association of the hOGG1 Ser326Cys polymorphism with sporadic amyotrophic lateral sclerosis

Amyotropic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease causing the loss of motoneurons of the brain and the spinal cord. The etiology of ALS is still uncertain, but males are at increased risk for the disease than females. Several studies have suggested that motoneurons in ALS might be subjected to the double insult of increased DNA oxidative damage and deficiencies in DNA repair systems. Particularly, increased levels of 8-oxoguanine and impairments of the DNA base excision repair system have been observed in neurons of ALS patients. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. To evaluate the role of the hOGG1 Ser326Cys polymorphism in sporadic ALS (sALS), we screened 136 patients and 129 matched controls. In the total population, we observed association between both the Cys326 allele (p=0.02) and the combined Ser326Cys+Cys326Cys genotype (OR=1.65, 95% CI=1.06-2.88) and increased risk of disease. After stratification by gender, the Cys326 allele (p=0.01), both the Ser326Cys genotype (OR=2.14, 95% CI=1.09-4.19) and the combined Ser326Cys+Cys326Cys genotype (OR=2.15, 95% CI=1.16-4.01) were associated with sALS risk only in males. No significant association between the Ser326Cys polymorphism and disease phenotype, including age and site of onset and disease progression, was observed. Present results suggest a possible involvement of the hOGG1 Ser326Cys polymorphism in sALS pathogenesis.

New findings in the ataxia of Charlevoix–Saguenay

The aim of the study was to enhance our understanding of the pathogenesis of the ataxia of Charlevoix–Saguenay, based on the findings presented herein. Five patients with a molecular diagnosis of this disease underwent clinical, radiological, ophthalmologic and electrophysiological examinations. Five novel mutations, which included nonsense and missense variants, were identified, with these resulting in milder phenotypes. In addition to the usual manifestations, a straight dorsal spine was found in every case, and imaging techniques showed loss of the dorsal kyphosis. Cranial MRI demonstrated hypointense linear striations at the pons. Tensor diffusion MRI sequences revealed that these striations corresponded with hyperplastic pontocerebellar fibres, and tractographic sequences showed interrupted pyramidal tracts at the pons. Ocular coherence tomography demonstrated abnormal thickness of the nerve fibre layer. Electrophysiological studies showed nerve conduction abnormalities compatible with a dysmyelinating neuropathy, with signs of chronic denervation in distal muscles. The authors suggest that the hyperplastic pontocerebellar fibres compress the pyramidal tracts at the pons, and that the amount of retinal fibres traversing the optic discs is enlarged. These facts point to the contribution of an abnormal developmental mechanism in the ataxia of Charlevoix–Saguenay. Accordingly, spasticity would be mediated by compression of the pyramidal tracts, neuromuscular symptoms by secondary axonal degeneration superimposed on the peripheral myelinopathy, while the cause of the progressive ataxia remains speculative. The distinctive aspect of the dorsal spine could be of help in the clinical diagnosis.