Mediterranean Diet and Cognitive Status in Free-Living Elderly: A Cross-Sectional Study in Northern Italy.

OBJECTIVE: Few data are available on the Italian elderly population with regard to adherence to the Mediterranean diet (MD) and cognitive impairment. Our aim was to investigate adherence to the MD and its association with cognitive function in an Italian urban sample. METHODS: A cross-sectional study of 279 participants aged ≥ 65 years (80 men, 199 women) was carried out at a nutritional center. Adherence to the MD was evaluated using a 14-item questionnaire. Cognitive function was assessed with the Mini-Mental State Examination (MMSE). RESULTS: The clinical and nutritional assessments performed revealed 30.1% to have a dietary pattern in accordance with the MD; 13.6% had suspected or mild cognitive impairment (MMSE score ≤ 23). The MD pattern was associated with a lower risk of cognitive impairment (odds ratio [OR] = 0.39; 95% confidence interval [CI], 0.15-0.99; p = 0.045), as was the consumption of wine (OR = 0.37; 95% CI, 0.16-0.84; p = 0.018) and nuts (OR = 0.30; 95% CI, 0.13-0.69, p = 0.005). No association was found with other food groups. CONCLUSION: A closer adherence to the MD was associated with a better cognitive status. Further cohort studies and randomized controlled trials are warranted.

Opposing roles of miR-21 and miR-29 in the progression of fibrosis in Duchenne muscular dystrophy.

Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-ß1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition.

Polysomnographic and neurometabolic features may mark preclinical autosomal dominant cerebellar ataxia, deafness, and narcolepsy due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1.

OBJECTIVE: We aimed to report the clinical picture of two asymptomatic daughters of a patient with autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN) due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1. METHODS: Clinical assessment based on history, neurologic examination, sleep recordings, neurophysiologic neuroimaging, and genetic tests was performed. RESULTS: History and neurologic examination in both subjects were unremarkable. Genetic analysis disclosed in both the paternally-inherited heterozygous point mutation in the DNMT1 gene. Sleep recordings found sleep-onset rapid eye movement periods (SOREMPs) and proton magnetic resonance spectroscopy (MRS) revealed increased cerebellar myoinositol (mI) in both subjects. Auditory and ophthalmologic investigations as well as structural brain magnetic resonance imaging (MRI) scans revealed no abnormalities. CONCLUSIONS: The two asymptomatic carriers of the heterozygous DNMT1 mutation for ADCA-DN, a late-onset neurodegenerative disease, presented with SOREMPs associated with an increase of mI in the brain, a marker of glial cell activity and density characteristic of early stages of neurodegenerative diseases. Therefore, SOREMPs may precede the clinical picture of ADCA-DN as an early polysomnographic marker of central nervous system involvement detected by MRS.

Narcolepsy is a common phenotype in HSAN IE and ADCA-DN.

We report on the extensive phenotypic characterization of five Italian patients from four unrelated families carrying dominant heterozygous DNMT1 mutations linked to two distinct autosomal dominant diseases: hereditary sensory and autonomic neuropathy with dementia and hearing loss type IE (HSAN IE) and autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Patients underwent genetic analysis of DNMT1 gene, neurophysiological tests investigating sleep, auditory functions and peripheral nervous system, ophthalmological studies including optical coherence tomography, lymphoscintigraphy, brain magnetic resonance and nuclear imaging, cerebrospinal fluid hypocretin-1, total tau, phosphorylated tau, amyloid-ß1-42 and 14-3-3 proteins measurement, skin, muscular and sural nerve biopsies. Exome and direct sequencing studies disclosed two different point mutations affecting exon 21 of DNMT1 gene in patients with ADCA-DN, a novel heterozygous point mutation in exon 20 in two affected HSAN IE siblings, and a trinucleotide deletion in exon 20 in the latter patient with HSAN IE. Phenotypic characterization pinpoints that ADCA-DN and HSAN IE represent two discrete clinical entities belonging to the same disease spectrum, with variable degree of overlap. Remarkably, narcolepsy with or without cataplexy with low/intermediate or normal cerebrospinal fluid hypocretin-1 is present in both diseases. The human leukocyte antigen DQB1*06:02 was absent in all patients. Other common symptoms and features observed in our cases, involving the central and peripheral nervous system, include deafness, optic neuropathy-previously not reported in HSAN IE-large and small fibres polyneuropathy and lower limbs oedema. Overall, the two syndromes share more characteristics than previously recognized and narcolepsy is common to both. HSAN IE and ADCA-DN are two extreme phenotypic manifestations of a DNMT1 methylopathy.

Mutations in DNMT1 cause autosomal dominant cerebellar ataxia, deafness and narcolepsy.

Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN) is characterized by late onset (30-40 years old) cerebellar ataxia, sensory neuronal deafness, narcolepsy-cataplexy and dementia. We performed exome sequencing in five individuals from three ADCA-DN kindreds and identified DNMT1 as the only gene with mutations found in all five affected individuals. Sanger sequencing confirmed the de novo mutation p.Ala570Val in one family, and showed co-segregation of p.Val606Phe and p.Ala570Val, with the ADCA-DN phenotype, in two other kindreds. An additional ADCA-DN kindred with a p.GLY605Ala mutation was subsequently identified. Narcolepsy and deafness were the first symptoms to appear in all pedigrees, followed by ataxia. DNMT1 is a widely expressed DNA methyltransferase maintaining methylation patterns in development, and mediating transcriptional repression by direct binding to HDAC2. It is also highly expressed in immune cells and required for the differentiation of CD4+ into T regulatory cells. Mutations in exon 20 of this gene were recently reported to cause hereditary sensory neuropathy with dementia and hearing loss (HSAN1). Our mutations are all located in exon 21 and in very close spatial proximity, suggesting distinct phenotypes depending on mutation location within this gene.

Identification of previously unreported mutations in CHRNA1, CHRNE and RAPSN genes in three unrelated Italian patients with congenital myasthenic syndromes.

Congenital myasthenic syndromes are rare genetic disorders compromising neuromuscular transmission. The defects are mainly mutations in the muscle acetylcholine receptor, or associated proteins rapsyn and Dok-7. We analyzed three unrelated Italian patients with typical clinical features of congenital myasthenic syndrome, who all benefitted from cholinesterase inhibitors. We found five mutations: a previously unreported homozygous alphaG378D mutation in the CHRNA1 gene, a previously unreported heterozygous epsilonY8X mutation associated with a known heterozygous epsilonM292del deletion in the CHRNE gene, and the common heterozygous N88K mutation associated with a previously unreported heterozygous IVS1 + 2T > G splice site mutation in the RAPSN gene. All three patients had two mutant alleles; parents or offspring with a single mutated allele were asymptomatic, thus all mutations exerted their effects recessively. The previously unreported mutations are likely to reduce the number of AChRs at the motor endplate, although the alphaG378D mutation might produce a mild fast channel syndrome. The alphaG378D mutation was recessive, but recessive CHRNA1 mutations have rarely been reported previously, so studies on the effect of this mutation at the cellular level would be of interest.

Concordance between severity of disease, disability and health-related quality of life in myasthenia gravis.

Aim of this study is to verify whether there is concordance between disease's severity, health-related quality of life (HRQoL) and disability in patients with myasthenia gravis (MG). 102 MG patients were clustered on the basis of HRQoL and disability scores into three groups: low disability and low HRQoL decrement (51 patients), intermediate disability and HRQoL decrement (28 patients), severe disability and high HRQoL decrement (23 patients). Cross tabulation with symmetric measures (Cramer's V and Contingency Coefficient) was used to verify the relationships between disease severity groups, based on the Myasthenia Gravis Foundation of America (MGFA) criteria, and obtained clusters. Results confirm a significant relationship between MG severity groups, HRQoL and disability profiles. In our opinion, HRQoL and disability instruments should be employed in clinical trials to match efficacy of treatments, measured on symptoms' reductions only, with their effects on patients' disability and HRQoL.

The relationship between health, disability and quality of life in myasthenia gravis: results from an Italian study.

Myasthenia gravis (MG) produces long term disability and affects health-related quality of life (HRQoL). This paper reports the relationship between HRQoL and disability in a group of patients with MG. Adult patients with MG were consecutively enrolled at the Neurological Institute "Carlo Besta". The World Health Organization Disability Assessment Schedule II (WHO-DAS II) and the Medical Outcome Survey 36-Item Short-Form Health Survey (SF-36) were employed, and non-parametric analysis (Spearman's rank correlation and Mann-Whitney U test) performed. One hundred and two consecutive adult patients with MG (70 female; mean age 47.2, sd 15.7) were recruited. The majority of WHO-DAS II and SF-36 scales were significantly correlated; WHO-DAS II summary score correlated better with SF-36 Physical Composite Score (PCS), than with mental composite score (MCS). Significant differences are also reported between patients with different muscle involvement in PCS and WHO-DAS II scores, while no difference was observed in MCS. The impact of MG on disability and HRQoL increases consistently with the disease's severity. Our study highlights that measurements of HRQoL and disability in patients with MG are correlated and sensitive enough to capture different clinical profiles' features. They measure different clinical and psychosocial facets, therefore we recommend employing specific assessments both for quality of life and disability in public health and clinical research on myasthenia gravis.

On the personal facets of quality of life in chronic neurological disorders.

Quality of life (QOL) is an important clinical endpoint, but it remarkably varies in patients with similar neurological conditions. This study explored the role of spirituality (i.e., the complex of personal transcendence, connectedness, purpose, and values) in determining QOL in chronic neurological disorders.~Seventy-two patients with epilepsy, brain tumours or ischemic or immune-mediate brain damage compiled inventories for QOL (WHOQOL 100), spirituality (Spiritual, Religious and Personal Beliefs, WHOSRPB), depression (Beck Depression Inventory, BDI), anxiety (State-Trait Anxiety Inventory, STAI), and cognitive self-efficacy (Multiple Ability Self-Report Questionnaire, MASQ) and underwent neuropsychological testing. With respect to 45 healthy controls, the patients reported worse QOL, with no difference between the four patient subgroups. Factor analyses of the WHOSRPB, STAI, and BDI scores and of the MASQ and neuropsychological test scores yielded four (Personal Meaning, Inner Energy, Awe and Openness, Mood) and three factors (Control Functions, Cognition, Memory), respectively. Mood, Cognition, Inner Energy, schooling, and subjective health status correlated with the WHOQOL scores, but at regression analysis only Mood and Inner Energy predicted QOL. This suggests that spirituality, as a personal dimension distinct from mood, contributes to determine QOL. A multidimensional assessment of QOL, including personal facets, may explain differences between patients with chronic neurological disorders.

Identification of international classification of functioning, disability and health relevant categories to describe functioning and disability of patients with myasthenia gravis.

PURPOSE: To describe functioning and health of patients with myasthenia gravis (MG) and to identify which are the most common problems patients encounter, by using the international classification of functioning, disability and health (ICF). METHOD: Adult patients with MG were recruited at C. Besta Neurological Institute. The ICF checklist was administered in individual sessions. Categories were identified as relevant if they were reported as a problem by more than 30% of patients (within activities and participation, the threshold was counted on capacity qualifier). RESULTS: One hundred two patients were enrolled (mean age 47.2; inpatients were 29.4%, females 68.6%) and 54 ICF categories were selected: 14 body functions categories (26% out of total selected categories), 2 body structures (4%), 22 activities and participation categories (41%) and 16 environmental factors (29%). Environmental factors were essentially reported as facilitators. CONCLUSIONS: Twelve ICF categories, not contained in ICF core-sets for neurological condition, related to mobility, household and labour activities were identified. The ICF categories identified in this study are an useful guideline for clinicians and researchers, for monitoring interventions and follow-up of clinical conditions on a broad set of functional areas, and for developing ICF-based assessment tools for patients with MG.

Disability and functional profiles of patients with myasthenia gravis measured with ICF classification.

The objective of this study is to describe functional profiles of patients with myasthenia gravis (MG), and the relationships among symptoms, activities and environmental factors (EF), by using WHO's International Classification of Functioning Disability and Health (ICF). Patients were consecutively enrolled at the Besta Institute of Milan, Italy. The ICF checklist was administered and two count-based indices were developed: 'extension', containing ICF categories rated with qualifiers 1-4, and 'severity', containing ICF categories rated with qualifiers 3-4. Spearman's correlation was performed to identify the relationships among symptoms, activities and EF, and linear regressions to identify the best predictors of performance indices in the activities and participation (A and P) domains. One hundred and one patients joined this study. Different values are reported in capacity and performance, the latter being 17.1% less limited in 'extension' and 13% in 'severity' index. Higher correlation indices are observed between A and P and body functions (BFs), than between A and P and EF. Problems in A and P are better explained by BF impairments than by facilitators within EF. In conclusion, EF plays a relevant role in improving MG patients' functioning. Nevertheless, difficulties in A and P, such as lifting objects or doing housework, are explained more by impairments in BF (e.g. in muscle endurance) than by the effect of EF (e.g. drugs and the support of family members). Methodologies and tools are needed to couple symptoms assessment with an evaluation of difficulties in executing activities and the environment's role, to plan appropriate interventions to meet MG patients' needs, especially in the labour sector, as 20% of patients gave up working before the retirement age.

Risk factors for tumor occurrence in patients with myasthenia gravis.

There is still uncertainty regarding risk factors for cancer occurrence in patients with myasthenia gravis (MG). The objective of this study is to determine the prevalence of extrathymic neoplasms in patients with MG and the factors associated with tumor occurrence. The archives of four tertiary MG centers were consulted and patients were interviewed on the main clinical features of the disease, the presence and type(s) of extrathymic neoplasms and other autoimmune disorders, and their symptomatic and immunosuppressant treatments (with detailed schedules). A retrospective cohort survey was undertaken comparing the demographic and clinical variables of patients with extrathymic neoplasms to those of the remaining MG population. 2,479 patients were traced and interviewed personally or through informants. The sample included 1,490 women and 989 men (mean age 54.7 years). Other autoimmune disorders were present in 216 cases (8.7%). Thymectomy was performed in 1,549 cases (62.5%), thymic hyperplasia and thymoma being the most common findings. Acetylcholinesterase-inhibitors were the most common treatment (93.5%), followed by steroids (64.3%), azathioprine (35.0%), plasma exchange (13.2%), immunoglobulins (7.5%), cyclosporine (5.3%), and cyclophosphamide (5.0%). 221 patients (8.9%) had one or more extrathymic tumors, 168 of which occurred after disease onset. Patients with and without extrathymic neoplasms were followed for 14.8 and 13.9 years, respectively. Variables shown by multivariate analysis to be associated with increased neoplastic risk included older age, thymoma and immunoglobulin use. Extrathymic tumors are a common finding in patients with MG and tend to be associated with age, thymoma, and immunoglobulin use.

Thymoma-associated myasthenia gravis: outcome, clinical and pathological correlations in 197 patients on a 20-year experience.

We studied 197 patients with thymoma-associated myasthenia gravis (T-MG) to identify variables that can influence the natural history of the disease and the therapeutical approaches. Multivariate analysis showed that neither clinical nor pathological variables were associated with a better chance to reach complete stable remission. The video-assisted thoracoscopic extended thymectomy (VATET) was not significantly correlated with a lower chance of achieving complete stable remission compared with the classical transsternal approach (T-3b) (p=0.1090). Thymoma recurrence was not correlated with surgery by VATET or T-3b. VATET was safe and reliable for removal of thymoma. The low chance of achieving remission (9.64%) in T-MG underlines the importance of an early diagnosis as well as the need for more aggressive therapeutic strategies.

Two cases of thymoma-associated myasthenia gravis without antibodies to the acetylcholine receptor.

Thymoma-associated myasthenia gravis is considered a more severe disease compared with non-thymomatous myasthenia gravis and is generally associated with antibodies to the acetylcholine receptor (AChR-Ab). Even though a single case of thymoma-associated myasthenia gravis with anti-muscle specific kinase (MuSK) antibodies has been reported, to our knowledge, seronegative thymoma-associated myasthenia gravis has not been described. We report on two cases of this disease without antibodies to AChR or MuSK as a further evidence of the variability of myasthenia gravis in terms of antibody profile and thymic pathological findings.

Immunomodulation of TGF-beta 1 in mdx mouse inhibits connective tissue proliferation in diaphragm but increases inflammatory response: implications for antifibrotic therapy.

Irreversible connective tissue proliferation in muscle is a pathological hallmark of Duchenne muscular dystrophy (DMD), a genetic degenerative muscle disease due to lack of the sarcolemmal protein dystrophin. Focal release of transforming growth factor-beta1 (TGF-beta1) is involved in fibrosis development. Murine muscular dystrophy (mdx) is genetically homologous to DMD and histopathological alterations comparable to those in DMD muscles occur in diaphragm of older mdx mice. To investigate the early development of fibrosis and TGF-beta1 involvement, we assessed diaphragms in 6-36-week-old mdx and C57/BL6 (control) mice for fibrosis, and used real-time PCR and ELISA to determine TGF-beta1 expression. Significantly greater fibrosis and TGF-beta1 expression were found in mdx from the 6th week. Mice treated with neutralizing antibody against TGF-beta1 had lower levels of TGF-beta1 protein, reduced fibrosis, unchanged muscles fiber degeneration/regeneration, but increased inflammatory cells (CD4+lymphocytes). These data demonstrate early and progressive fibrosis in mdx diaphragm accompanied by TGF-beta1 upregulation. Reduction of TGF-beta1 appears promising as a therapeutic approach to muscle fibrosis, but further studies are required to evaluate long term effects of TGF-beta1 immunomodulation on the immune system.

Increased toll-like receptor 4 expression in thymus of myasthenic patients with thymitis and thymic involution.

Thymic abnormalities are present in approximately 80% of myasthenia gravis (MG) patients, and the thymus seems to be the main site of autosensitization to the acetylcholine receptor. In view of findings that the innate immune system can generate an autoimmune response, we studied the expression of Toll-like receptors (TLRs) 2 to 5, key components of innate immunity signaling pathways, in 37 thymuses from patients with autoimmune MG. TLR4 mRNA levels were significantly greater in thymitis (hyperplasia with diffuse B-cell infiltration) and involuted thymus than in germinal center hyperplasia and thymoma. By immunohistochemistry and confocal microscopy, cells positive for TLR4 protein were rarely detected in thymoma. However, in thymitis TLR4 protein was mostly found on epitheliomorphic (cytokeratin-positive) cells located in close association with clusters of acetylcholine receptor-positive myoid cells in thymic medulla and also at the borders between cortical and medullary areas. B cells were never TLR4-positive. TLR4 protein was also present in remnant tissue of involuted thymus. This is the first finding of a possible link between innate immunity and MG. We speculate that in a subgroup of MG patients, an exogenous or endogenous danger signal may activate the innate immune system and give rise to TLR4-mediated mechanisms contributing to autoimmunity.

Abnormal lysosomal and ubiquitin-proteasome pathways in 19p13.3 distal myopathy.

We describe a second large Italian kindred with autosomal dominant vacuolar myopathy characterized by variable severity, adult-onset weakness of distal limb muscles, and no cardiac involvement. At least 19 individuals over four generations are affected. Histopathological and immunochemical features of the vacuoles, present in many fibers, indicate protein degradation abnormalities with dysregulation of the lysosomal pathway and activation of the ubiquitin-proteasomal pathway. Linkage analysis localized the defect to the 19p13.3 locus in a region with no known genes. We speculate that the primary defect may be an abnormality in the lysosomal degradation pathway or related components.

Anti-MOG autoantibodies in Italian multiple sclerosis patients: specificity, sensitivity and clinical association.

There is considerable evidence that multiple sclerosis (MS) is an immune-mediated disease characterized by infiltration of inflammatory cells into the CNS and demyelination. Several myelin proteins may be encephalitogenic, including myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein (MOG), the latter being expressed on the external layer of myelin sheaths and hence accessible to antibody attack. We investigated MOG autoreactivity in serum and cerebrospinal fluid (CSF) by ELISA, employing the recombinant extracellular domain of MOG as antigen. We tested serum samples from 262 MS patients (175 relapsing-remitting, 43 primary progressive and 44 secondary progressive), 131 patients with other neurological diseases (OND) and 307 healthy controls. No patients or controls were receiving immunomodulating treatments. We found anti-MOG antibodies in the serum of 13.7% MS patients, mainly in those with secondary progressive MS (25%), in 13.7% of OND patients and in 6.2% of controls. We found a direct correlation (R(2) = 0.6, P = 0.002) between disease severity and anti-MOG titer only in patients with primary and secondary progressive MS. Anti-MOG antibodies were present in the CSF of 11.4% MS patients and 18.9% OND patients. Intrathecal synthesis of anti-MOG antibodies was demonstrated in four (4.5%) of MS patients and no OND patients. Anti-MOG antibodies are not specific for MS; however, they may characterize a subset of MS patients and this may be revealed by serial assays in relation to changing disease phase.

Breakdown of tolerance to a self-peptide of acetylcholine receptor alpha-subunit induces experimental myasthenia gravis in rats.

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97-116 of the AChR alpha subunit. A proliferative T cell response against the corresponding rat sequence (R97-116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97-116 or T97-116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97-116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.