Migraine as possible red flag of PFO presence in suspected demyelinating disease.

OBJECTIVES: To investigate a possible association between isolated white matter lesions suggestive of demyelinating disease in magnetic resonance imaging (MRI) and patent foramen ovale (PFO) evidence in migraine patients, with or without aura. MATERIALS: 31 migraine patients, 28 females and 3 males, with MRI evidence of white matter lesions suggestive of demyelinating disease according to the Barkhof Criteria. All patients underwent further diagnostics including lumbar puncture, autoimmunity panel and cardiological evaluation to detect the presence of PFO. The mean duration of follow-up was 3.46 years and MIPAV software was used to analyze MRI imaging. RESULTS: 14 of the 31 patients (45%) had PFO. A significant association was found between PFO and migraine with visual aura (p < 0.001). No difference in lesion number, volume or area between patients with and without PFO was found, but the distribution was mainly occipital (p < 0.001) in patients with PFO. The follow-up showed a stationary lesion load in all PFO patients; no infratentorial or spinal cord lesion and no enhancement or corpus callosum lesion was ever detected. At the end of follow-up four patients developed multiple sclerosis: younger age at first MRI and oligoclonal bands were associated risk factors. CONCLUSIONS: Migraine is often one of the main symptoms leading to MRI, and in many cases white matter lesions of unspecific significance are discovered, thus placing demyelinating diseases in the differential diagnosis. Our study underlines the potential pathogenetic role of PFO in generating white matter lesions in migraine patients (45%), particularly those with visual aura and occipital lesions. For this reason, we affirm that PFO represents a cardinal point in the differential diagnosis of suspected demyelinating disease.

Nano-delivery systems for encapsulation of dietary polyphenols: An experimental approach for neurodegenerative diseases and brain tumors.

Neurodegenerative diseases (NDs) and brain tumors are severe, disabling, and incurable disorders that represent a critical problem regarding human suffering and the economic burden on the healthcare system. Because of the lack of effective therapies to treat NDs and brain tumors, the challenge for physicians is to discover new drugs to improve their patients' quality of life. In addition to risk factors such as genetics and environmental influences, increased cellular oxidative stress has been reported as one of the potential common etiologies in both disorders. Given their antioxidant and anti-inflammatory potential, dietary polyphenols are considered to be one of the most bioactive natural agents in chronic disease prevention and treatment. Despite the protective activity of polyphenols, their inefficient delivery systems and poor bioavailability strongly limit their use in medicine and functional food. A potential solution lies in polymeric nanoparticle-based polyphenol delivery systems that are able to enhance their absorption across the gastrointestinal tract, improve their bioavailability, and transport them to target organs. In the present manuscript, we provide an overview of the primary polyphenols used for ND and brain tumor prevention and treatment by focusing on recent findings, the principal factors limiting their application in clinical practice, and a promising delivery strategy to improve their bioavailability.

Autosomal-dominant myopia associated to a novel P4HA2 missense variant and defective collagen hydroxylation.

We recently described a complex multisystem syndrome in which mild-moderate myopia segregated as an independent trait. A plethora of genes has been related to sporadic and familial myopia. More recently, in Chinese patients severe myopia (MYP25, OMIM:617238) has been linked to mutations in P4HA2 gene. Seven family members complaining of reduced distance vision especially at dusk underwent complete ophthalmological examination. Whole-exome sequencing was performed to identify the gene responsible for myopia in the pedigree. Moderate myopia was diagnosed in the family which was associated to the novel missense variant c.1147A > G p.(Lys383Glu) in the prolyl 4-hydroxylase,alpha-polypeptide 2 (P4HA2) gene, which catalyzes the formation of 4-hydroxyproline residues in the collagen strands. In vitro studies demonstrated P4HA2 mRNA and protein reduced expression level as well as decreased collagen hydroxylation and deposition in mutated fibroblast primary cultures compared to healthy cell lines. This study suggests that P4HA2 mutations may lead to myopic axial elongation of eyeball as a consequence of quantitative and structural alterations of collagen. This is the first confirmatory study which associates a novel dominant missense variant in P4HA2 with myopia in Caucasian patients. Further studies in larger cohorts are advisable to fully clarify genotype-phenotype correlations.

Multiple spinal nerve enlargement and SOS1 mutation: Further evidence of overlap between neurofibromatosis type 1 and Noonan phenotype.

Neurofibromatosis type 1 (NF1) has long been considered a well-defined, recognizable monogenic disorder, with neurofibromas constituting a pathognomonic sign. This dogma has been challenged by recent descriptions of patients with enlarged nerves or paraspinal tumors, suggesting that neurogenic tumors and hypertrophic neuropathy may be a complication of Noonan syndrome with multiple lentigines (NSML) or RASopathy phenotype. We describe a 15-year-old boy, whose mother previously received clinical diagnosis of NF1 due to presence of bilateral cervical and lumbar spinal lesions resembling plexiform neurofibromas and features suggestive of NS. NF1 molecular analysis was negative in the mother. The boy presented with Noonan features, multiple lentigines and pectus excavatum. Next-generation sequencing analysis of all RASopathy genes identified p.Ser548Arg missense mutation in SOS1 in the boy, confirmed in his mother. Brain and spinal magnetic resonance imaging scans were negative in the boy. No heart involvement or deafness was observed in proband or mother. This is the first report of a SOS1 mutation associated with hypertrophic neuropathy resembling plexiform neurofibromas, a rare complication in Noonan phenotypes with mutations in RASopathy genes. Our results highlight the overlap between RASopathies, suggesting that NF1 diagnostic criteria need rethinking. Genetic analysis of RASopathy genes should be considered when diagnosis is uncertain.

Efficient cultivation of neural stem cells with controlled delivery of FGF-2.

Neural stem cells (NSCs) raised the hope for cell-based therapies in human neurodevelopmental and neurodegenerative diseases. Current research strategies aim to isolate, enrich, and propagate homogeneous populations of neural stem cells. Unfortunately, several concerns with NSC cultures currently may limit their therapeutic promise. Exhaustion of growth factors and/or their uncontrolled release with burst and fall in their concentration may greatly affect the in vitro behavior of NSCs. In this context, we investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus improve in vitro NSC cultivation. We demonstrated that NSCs cultivated in media with a controlled release of FGF-2 from a polyelectrolyte polymer showed a higher proliferation rate, and reduced apoptosis and senescence. In these experimental conditions NSCs preserve their stemness properties for a longer period of time compared with controls. Also of interest is that cell fate properties are conserved as well. The controlled release of FGF-2 reduced the level of oxidative stress and this is associated with a lower level of damaged DNA. This result may explain the reduced level of senescence and apoptosis in NSCs cultivated in the presence of hydrogel-releasing FGF-2.

Neurofibromatosis type 1 and optic pathway glioma. A long-term follow-up.

AIM: Optic pathway gliomas (OPG) are the predominant intracranial tumours associated with neurofibromatosis type 1 (NF1). The aim of this study was to evaluate the prevalence and the outcome of OPG in 200 NF1 patients (122 males and 78 females, aged 1-25 years) followed up to 16 years (mean of 6 years). METHODS: All children were evaluated by a detailed physical, neurological and ophthalmological examination. Fifteen out of 200 (7.5%) of these patients (7 males, 8 females) were identified with evidence of optic pathway tumours. RESULTS: Nine children had symptoms such as endocranial hypertension, seizures, headache; 4 patients only showed anomalies at ophthalmological examination; 2 patients had no symptoms or signs. All children had evidence of optic pathway tumour on magnetic resonance imaging. Three had a prechiasmal tumour, 2 had a chiasmal tumour, 1 had prechiasmal/chiasmal tumour, 2 had a prechiasmal/chiasmal and postchiasmal tumour, 2 had a chiasmal and postchiasmal tumour, 4 had a massive involvement of the optic system, 1 child exhibited a bilateral involvement of the optic nerves with additional impairment of the chiasm. Four patients had partial and/or subtotal spontaneous regression. CONCLUSIONS: Because optic pathway tumours arise in children younger than 6 years of age, all NF1 children should undergo yearly ophtalmologic examination and growth assessment to monitor signs of precocious puberty.

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1.

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of beta-oxidation flux by labeled palmitate demonstrates that tBid inhibits beta-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on beta-oxidation. The unexpected role of tBid in the regulation of lipid beta-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

RB and RB2/p130 genes demonstrate both specific and overlapping functions during the early steps of in vitro neural differentiation of marrow stromal stem cells.

Marrow stromal stem cells (MSCs) are stem-like cells that are currently being tested for their potential use in cell therapy for a number of human diseases. MSCs can differentiate into both mesenchymal and nonmesenchymal lineages. In fact, in addition to bone, cartilage and fat, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes. RB and RB2/p130 genes are involved in the differentiation of several systems. For this reason, we evaluated the role of RB and RB2/p130 in the differentiation and apoptosis of MSCs under experimental conditions that allow for MSC differentiation toward the neuron-like phenotype. To this end, we ectopically expressed either RB or RB2/p130 and monitored proliferation, differentiation and apoptosis in rat primary MSC cultures induced to differentiate toward the neuron-like phenotype. Both RB and RB2/P130 decreased cell proliferation rate. In pRb-overexpressing cells, the arrest of cell growth was also observed in the presence of the HDAC-inhibitor TSA, suggesting that its antiproliferative activity does not rely upon the HDAC pathway, while the addition of TSA to pRb2/p130-overexpressing cells relieved growth inhibition. TUNEL reactions and studies on the expression of genes belonging to the Bcl-2 family showed that while RB protected differentiating MSCs from apoptosis, RB2/p130 induced an increase of apoptosis compared to controls. The effects of both RB and RB2/p130 on programmed cell death appeared to be HDAC- independent. Molecular analysis of neural differentiation markers and immunocytochemistry revealed that RB2/p130 contributes mainly to the induction of generic neural properties and RB triggers cholinergic differentiation. Moreover, the differentiation potentials of RB2/p130 and RB appear to rely, at least in part, on the activity of HDACs.

Charcot-Marie-Tooth disease with giant axons: a clinicopathological and genetic entity.

The authors report an Italian family with autosomal-dominant Charcot-Marie-Tooth disease (CMT) in which there were giant axons in the sural nerve biopsy. Linkage to the known CMT2 loci (CMT2A, CMT2B, CMT2D, CMT2F) and mutations in the known CMT2 genes (Cx32, MPZ, NEFL), GAN, NEFM, and CMT1A duplication/HNPP deletion were excluded. This family with CMT and giant axons has a pathologic and genetic entity distinct from classic CMT.

EGF-responsive rat neural stem cells: molecular follow-up of neuron and astrocyte differentiation in vitro.

Neural stem cells (NSCs) could be very useful for the "cell therapy" treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF-responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF-containing medium, cells were plated on poly-L-ornithine-coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the "p53 network," was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.

RB2/p130 ectopic gene expression in neuroblastoma stem cells: evidence of cell-fate restriction and induction of differentiation.

The activity of the RB2/p130 gene, which is a member of the retinoblastoma gene family, is cell-cycle-regulated and plays a key role in growth inhibition and differentiation. We used neuroblastoma cell lines as a model for studies on neural crest progenitor cell differentiation. We show that Rb2/p130 ectopic protein expression induces morphological and molecular modifications, promoting differentiation of intermediate (I) phenotype SK-N-BE(2)-C neuroblastoma cells towards a neuroblastic (N) rather than a Schwann/glial/melanocytic (S) phenotype. These modifications are stable as they persist even after treatment with an S-phenotype inducer. Rb2/p130 ectopic expression also induces a more differentiated phenotype in N-type SH-SY-5Y cells. Further, this function appears to be independent of cell-cycle withdrawal. The data reported suggest that the Rb2/p130 protein is able to induce neuronal lineage specification and differentiation in neural crest stem and committed neuroblastoma cells, respectively. Thus, the Rb2/p130 protein seems to be required throughout the full neural maturation process.

pRb2/p130 gene overexpression induces astrocyte differentiation.

There are many data on the activity of the RB gene in neural differentiation and apoptosis, but the role of pRb2/p130 in neuronal and glial maturation has been far less investigated. To elucidate the role of pRb2/p130 in astrocyte development we overexpressed this protein in astrocytoma and normal astrocyte cultures by adenoviral-mediated gene transfer. In astrocytoma cells, p130/RB2 overexpression resulted in a significant reduction of cell growth and in an increased G(0)/G(1) cell population. We did not observe any induction of programmed cell death as determined by TUNEL reaction. Interestingly, pRb2/p130 overexpression induced astrocyte differentiation. Astrocyte cell cycle arrest and differentiation seemed to proceed through a way distinct from the p53 pathway.

17-beta estradiol elicits an autocrine leiomyoma cell proliferation: evidence for a stimulation of protein kinase-dependent pathway.

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2-dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP-kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2-stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI-3-K, and PLCgamma, and the concomitant activation of ancillary protein kinases, are related to E2-induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti-PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2-induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down-regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation.

Increased expression of IGF-binding protein-5 in Duchenne muscular dystrophy (DMD) fibroblasts correlates with the fibroblast-induced downregulation of DMD myoblast growth: an in vitro analysis.

In DMD the progressive loss of muscle ability and concomitant increasing fibrosis might originate from, besides other causes, the fibroblast paracrine inhibition of satellite cell "growth." In this study we report that in myoblast/fibroblast coculture experiments, the presence of DMD fibroblasts negatively interfered with DMD myoblast growth to an extent directly proportional to the percentage of DMD fibroblasts present in the mixed-cell cultures. Moreover, the observation that media conditioned with proliferating DMD fibroblasts inhibited the growth of DMD myoblasts more seriously than did control fibroblast-conditioned media suggested a paracrine effect by diffusible factors. IGF-binding proteins could act as such diffusible factors; in fact, IGFBP-5 transcript increased threefold in DMD fibroblasts proliferating in DMD muscle extracts, whereas IGFBP-3 mRNA decreased. In addition, high levels of IGFBP-5 protein were detected in DMD fibroblast-conditioned media. In neutralizing IGFBP-5 in DMD fibroblast-conditioned media by means of specific antibodies, or inhibiting IGFBP-5 gene expression in DMD fibroblasts by means of oligo antisense, the fibroblast-conditioned media lost inhibitory power over DMD myoblast proliferation.

Defective growth in vitro of Duchenne Muscular Dystrophy myoblasts: the molecular and biochemical basis.

As the molecular basis of Duchenne Muscular Dystrophy (DMD) was being discovered, increasing focus was placed on the mechanisms of progressive failure of myoregeneration. In this study, we propose a pathogenesis model for DMD, where an autocrine growth factor release of TGF-beta1-from necrotic myofibers-could contribute to the increasing loss of muscle regeneration. In fact, we report evidence that DMD myoblasts reduce their proliferation rate, in time and later cultures; in connection with this, we observed TGF-beta1 increase in conditioned media of DMD myoblasts, able to control the myoblast growth by reducing fusion and differentiation of DMD satellite cells.

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc-interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G(0)/G(1) cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell.

Antisense inhibitory effect: a comparison between 3'-partial and full phosphorothioate antisense oligonucleotides.

Phosphorothioate (PS) antisense oligonucleotides are currently used to inhibit many cell functions both in vivo and in vitro. However, these modified oligos provide reasonable sequence specificity only within a narrow concentration range. To overcome such a limitation we synthesized antisense oligomers, partially phosphorothioated, targeted against the human N-myc mRNA. We utilized such modified oligomers in a human neuroblastoma cell line where the N-myc gene expression was very high, and compared them to full phosphorothioate oligonucleotides. Both full PS and partial PS antisense oligos produced a maximum reduction in target mRNA after 6 h of treatment. They were able to maintain a good level of inhibition for 20 h only at high concentration. While partial PS oligos produced a dose dependent and sequence specific inhibition of N-myc mRNA, full PS molecules suffer from some disadvantages at the highest concentration used. Our results showed that partial PS molecules were capable of reducing gene expression showing a greater sequence specificity over a far broader concentration range. For this reason we conclude that partial PS antisense oligos, with respect to full PS antisense oligos, might be particularly useful for studying gene function.

Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product.

To clarify the role and function of the N-myc product in cell differentiation and apoptosis, we used the antisense oligonucleotide technique to inhibit N-myc gene expression in neuroblastoma cells with different phenotypes: intermediate (I) and neuronal (N), or Schwann-glia (S), respectively. The results suggest that N-myc operates along different pathways. Inhibiting N-myc gene expression either results in suppression of cell proliferation or in induction of differentiation and/or apoptosis.

Modulation of in vitro myogenesis induced by different polymer substrates.

The understanding of substrate dependence of cellular differentiation is important in the surface design of biocompatible artificial devices as well as cell-incorporated tissue engineered devices. In an attempt to understand some of the genetic and epigenetic aspects of the control of cell differentiation in the presence of two different materials, Chronoflex (CH) and plasma treated Chronoflex coated with Hyaluronan (CH-HA), we used primary cultures of human myogenic cells, a model that encompasses cell proliferation, migration, fusion, and differentiation dependent gene activation. By testing both the material samples on the growth of human myoblasts in primary cultures, we demonstrated that both CH and CH-HA substrates were able to support the cell growth since they did not affect cell count and DNA synthesis. On the contrary, the degree of myoblast differentiation, assessed as a function of creatine phosphokinase (CPK) activity on living cells, was completely different on the two biomaterials. Indeed, the amount of CPK increased on CH-HA cultured cells as a result of myotube formation, while CH grown myoblasts remained unfused and displayed no increase on the CPK activity even after 12 days culture. Moreover, the expression level of MyoD and myogenin mRNA, both related to myogenic cell differentiation, appeared extremely low in CH-grown cells, while they were rapidly induced in CH-HA cultured myoblasts.