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Lodola F.,Molecular Cardiology | Morone D.,Humanitas Research Hospital | Denegri M.,Molecular Cardiology | Bongianino R.,Molecular Cardiology | And 15 more authors.
Cell Death and Disease | Year: 2016

Catecholaminergic Polymorphic Ventricular Tachycardia type 2 (CPVT2) is a highly lethal recessive arrhythmogenic disease caused by mutations in the calsequestrin-2 (CASQ2) gene. We have previously demonstrated that viral transfer of the wild-type (WT) CASQ2 gene prevents the development of CPVT2 in a genetically induced mouse model of the disease homozygous carrier of the R33Q mutation. In the present study, we investigated the efficacy of the virally mediated gene therapy in cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs) obtained from a patient carrying the homozygous CASQ2-G112+5X mutation. To this end, we infected cells with an Adeno-Associated Viral vector serotype 9 (AAV9) encoding the human CASQ2 gene (AAV9-hCASQ2). Administration of the human WT CASQ2 gene was capable and sufficient to restore the physiological expression of calsequestrin-2 protein and to rescue functional defects of the patient-specific iPSC-derived CMs. Indeed, after viral gene transfer, we observed a remarkable decrease in the percentage of delayed afterdepolarizations (DADs) developed by the diseased CMs upon adrenergic stimulation, the calcium transient amplitude was re-established and the density and duration of calcium sparks were normalized. We therefore demonstrate the efficacy of the AAV9-mediated gene replacement therapy for CPVT2 in a human cardiac-specific model system, supporting the view that the gene-therapy tested is curative in models with different human mutations of CPVT.


Buonocore M.,Unit of Clinical Neurophysiology and Neurodiagnostic Skin Biopsy | Aloisi A.M.,Unit of Pain Medicine IRCCS Fondazione Salvatore Maugeri Pavia | Aloisi A.M.,University of Siena | Barbieri M.,Unit of Pain Medicine IRCCS Fondazione Salvatore Maugeri Pavia | And 2 more authors.
Journal of Cellular Physiology | Year: 2010

Vertebral fractures often cause intractable pain. To define the involvement of vertebral body innervation in pain, we collected specimens from male and female patients during percutaneous kyphoplasty, a procedure used for reconstruction of the vertebral body. Specimens were taken from 31 patients (9 men and 22 women) suffering high-intensity pain before surgery. In total, 1,876 histological preparations were obtained and analysed. Immunohistochemical techniques were used to locate the nerves in the specimens. The nerve fibres were labelled by indirect immunofluorescence with the primary antibody directed against Protein Gene Product 9.5 (PGP 9.5), a pan-neuronal marker; another primary antibody directed against type IV collagen (Col IV) was used to identify vessels and to determine their relationship with vertebral nerve fibres. The mean percentage of samples in which it was possible to identify nerve fibres was 35% in men and 29% in women. The percentages varied depending on the spinal level considered and the sex of the subject, nerve fibres being mostly present around vessels (95%). In conclusion, there is scarce innervation of the vertebral bodies, with a clear prevalence of fibres located around vessels. It seems unlikely that this pattern of vertebral body innervation is involved in vertebral pain or in pain relief following kyphoplasty. © 2009 Wiley-Liss, Inc.


Di Pasquale E.,National Research Council Italy | Lodola F.,Molecular Cardiology | Miragoli M.,University of Milan | Miragoli M.,University of Parma | And 13 more authors.
Cell Death and Disease | Year: 2013

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after b-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4- chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca2+/calmodulin-dependent serinethreonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies. © 2013 Macmillan Publishers Limited. All rights reserved.


Buonocore M.,Unit of Clinical Neurophysiology and Neurodiagnostic Skin Biopsy | Demartini L.,Unit of Pain Medicine
Neuromodulation | Year: 2016

Background: Although the number of patients with chronic neuropathic pain treated by spinal cord stimulation (SCS) is continuously increasing, its analgesic mechanism remains to be elucidated. Previous studies have demonstrated that classical SCS (low stimulation frequency evoking paresthesia) inhibits the somatosensory evoked potentials (SEPs). Case Presentation: We describe here the results of a series of SEPs recordings performed in a female patient with chronic pain, using four different types of SCS: the classical SCS (60 Hz, 250 μsec) and three paresthesia free SCS modalities: high frequency (10 kHz, 20 μsec) and two types of high-density SCS (500 Hz, 500 μsec and 200 Hz, 1000 μsec). All the tested SCS modalities completely inhibited the SEPs cortical responses, with an immediate recovery of the inhibition after turning the stimulator off. Conclusions: All the tested SCS modalities are able to inhibit SEPs and thus the lemniscal system. In particular, both paresthesia and paresthesia free SCS affect SEPs in the same manner. The presence of this inhibitory effect during paresthesia free modalities suggests that it is independent from the generation of action potentials, with a probable mechanism acting at the stimulation site. Further studies investigating the relationship between the inhibition of the lemniscal system and the analgesic effect of the SCS are, therefore, warranted. © 2016 International Neuromodulation Society.


Magri F.,University of Pavia | Buonocore M.,Unit of Clinical Neurophysiology and Neurodiagnostic Skin Biopsy | Oliviero A.,Hospital Nacional Of Paraplejicos | Rotondi M.,University of Pavia | And 4 more authors.
European Journal of Endocrinology | Year: 2010

Aim: To evaluate, by using skin biopsy technique, the intraepidermal nerve fiber (IENF) density in a group of untreated patients with hypothyroidism, either overt (OH) or subclinical (SH), who did not complain of neurologic symptoms. Methods: We evaluated 18 neurologically asymptomatic patients newly diagnosed with OH or SH. Fifteen healthy, age-matched, controls were also studied. A nerve conduction study was performed. Skin biopsy was carried out from the skin of upper thigh and distal leg. Nerve fiber density was measured using an immunofluorescence technique. The density of innervation was calculated by counting only fibers crossing the basement membrane. Results: Electroneurographic parameters were similar in patients and controls. When compared with healthy controls, patients with OH or SH showed a significantly lower IENF density. As assessed by the proximal/distal fiber density ratio, the hypothyroid neuropathy was length dependent. When individually considered, an abnormally reduced IENF was observed in 60% of patients with OH at the distal leg and in 20% at the proximal site. In patients with SH, an abnormal IENF density was found at the distal leg in 25% of cases and at the proximal thigh in 12.5% of cases. Conclusions: Our study provides the first direct demonstration of reduced IENF density in patients with OH or SH. In all patients, the IENF density reduction was length dependent. These findings suggest that a considerable number of untreated hypothyroid patients may have preclinical asymptomatic small-fiber sensory neuropathy. © 2010 European Society of Endocrinology.


Buonocore M.,Unit of Clinical Neurophysiology and Neurodiagnostic Skin Biopsy | Buonocore M.,Center for Research in Pain Pathophysiology and Treatment | Gatti A.M.,Unit of Clinical Neurophysiology and Neurodiagnostic Skin Biopsy | Amato G.,Unit of Clinical Neurophysiology and Neurodiagnostic Skin Biopsy | And 3 more authors.
Journal of Cellular Physiology | Year: 2012

Most post-herpetic neuralgia (PHN) patients suffer from tactile allodynia (pain evoked by lightly touching the skin) and it is frequently the dominant clinical manifestation. The pathophysiology of tactile allodynia in PHN patients is poorly understood and this is one of the major limits to the development of appropriate therapies. Epidermal nerve fibres (ENFs) are free nerve endings of small-diameter A-delta and C primary afferents, which can easily be assessed by neurodiagnostic skin biopsy (NSB). The aim of this study was to establish the correlation between the residual epidermal innervation of the allodynic skin and the intensity of tactile allodynia in that area. Twenty-five patients (13 males and 12 females) with PHN were enrolled. Eighteen patients had PHN in the thoracic dermatome, four in the cervical, two in the trigeminal and one in the lumbar. The severity of allodynia evoked by a paintbrush was graded according to an eleven-point numerical scale. A skin biopsy was obtained from the maximal allodynia area and from the contralateral skin. Nerve fibres were labelled with indirect immunofluorescence. Results showed that epidermal innervation was lower in the allodynic skin than in the contralateral skin, although there was great variability among patients. There was no correlation between severity of allodynia and epidermal innervation of the PHN skin. In conclusion, the present study further indicates peripheral nervous system involvement in PHN but does not support a direct correlation between epidermal innervation changes and tactile allodynia. © 2011 Wiley Periodicals, Inc.

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