Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.

REASONS FOR PERFORMING STUDY: Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. OBJECTIVES: The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. STUDY DESIGN: Randomised in vivo experiment. METHODS: We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. RESULTS: Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken ß-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. CONCLUSIONS: The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the distribution pattern of transduction.

Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain.

Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog's lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Myopathy as the first symptom of hypokalemic periodic paralysis--case report of a girl from a Polish family with CACNA1S (R1239G) mutation.

PURPOSE: Presenting the case of unusual onset hypokalemic periodic paralysis (HypoPP) where myopathy had developed two years before paralysis occurred. MATERIAL AND METHODS: A Polish three-generation family with HypoPP and mutation in CACNA1S (R1239G) has been investigated. Clinical presentation with unusual onset of the disease, biopsy results and genetic research in one family member were described. CONCLUSION: HypoPP is a rare disease it needs to be taken into consideration not only in cases of paroxysmal weakness but also when there is myopathy of unknown origin.

Bilateral facial nerve palsy in the course of neuroborreliosis in children-dynamics, laboratory tests and treatment.

PURPOSE: Presentation of four patients with bilateral peripheral facial nerve palsy as a clinical manifestation of neuroborreliosis in children--diagnostic, treatment and prognosis. MATERIAL AND METHODS: In 2002-2004 in The Chair and Department of Developmental Neurology, 24 children from the Wielkopolska region were admitted with diagnosis of borreliosis. Among all the children with borreliosis, confirmed by serologic examination, 4 (16.7%) demonstrated bilateral peripheral facial palsy (PFP). We investigated the presence of IgM class and IgG class specific antibodies in the sera and cerebrospinal fluid (CSF) of 4 patients with bilateral PFP. (Detected by immunoenzymatic methods--ELISA.) RESULTS: Before the occurrence of PFP all the children manifested unspecified systemic symptoms such as headaches, muscle and articulation pains, weakness and in two cases a mood depression. At first all patients demonstrated elevated IgM antibodies and proper levels of IgG antibodies. Control tests administered within 2-14 months later reduction of antibodies was indicated. Two patients demonstrated significant pleocytosis in CSF test, (without the meningeal symptoms). All children were treated with physiotherapeutic procedures and were administered antibiotic intravenously. CONCLUSIONS: PFP is one of the most frequent neurological symptoms of borreliosis in children. In case of acute PFP and especially the bilateral form of PFP, neuroborreliosis is the most probable diagnosis. All children reported PFP at one side first and after several weeks the paresis of the facial nerve on the opposite side usually appeared. The clinical state of children started to improve after the introduction of physiotherapy and this process usually lasted several months.

[Diagnostic value of sleep deprivation as the EEG activation method in diagnosis of epilepsy in developmental age]. / Wartosc diagnostyczna deprywacji snu jako metody aktywacji EEG w rozpoznawaniu padaczki w wieku rozwojowym.

The paper presented the significance of EEG activation in diagnosing epilepsy in children and adolescents. EEG records of 108 patients between 3-19 years of age with the occurrence of various types of paroxysmal disorders who were admitted to Chair and Department of Developmental Neurology University of Medical Sciences in Poznan were analysed. Standard EEG was administered to all children twice: the first examination at rest with hyperventilation (HV) and photostimulation (FS) and the second one after total or partial sleep deprivation (DS). At awaking state normal records were stated in 57% of cases, in 15% paroxysmal changes in HV or FS appeared, in the rest of cases (28%) generalised paroxysmal disorders, localised changes, lateralised or diffused were noted. Among EEG activation methods DS occurred to be the most effective after it, 52 patients (48%) were diagnosed for generalised paroxysmal changes, 20% for localised, 3% lateralised and for 6% for diffused changes. Normal EEG were recorded in 22% of examined children and adolescents. The epilepsy were diagnosed in 59 (55%) of patients mostly with generalised seizures (28) partial secondarily generalised (16) and partially complex (11). In 49 (45%) children were diagnosed with other paroxysmal disorders.

[The us of video-EEG in the diagnosis of epilepsy syndromes in the developmental age]. / Zastosowanie wideo-EEG w diagnostyce zespolów padaczkowych wieku rozwojowego.

In the Department of Developmental Neurology, K. Marcinkowski University of Medical Sciences in Poznan 120 video-EEG were performed in 1997-1998. Video-EEG monitoring was executed by means of computer electroencephalograph "Ceegraph" (Biologic--USA). A group of 20 patients between the age of 20 months up to 19 years, whose video-EEG was analysed, was hospitalised because of diagnostic doubtful seizures. Stimultaneous EEG and clinical events recordings were evaluated with the use of videotape at all children. In the investigated group, 13 patients with epileptic seizures (5 with nonepileptic seizures, 1 patient with choreatic movements and 1 with nocturnal myoclonus) were recorded. There were 3 children classified with generalised absence seizures, 3 with partial seizures and 1 patient with myoclonic seizure among the children with epilepsy. Moreover, there were several different cases diagnosed: 3 patients presented Lennox-Gastaut syndrome, 1 was classified with West's syndrome, 1 patient was characterised by Alpers syndrome and 1 with tuberous sclerosis. Psychogenic pseudoepileptic seizures were classified in 5 patients, in 3 of them both epileptic and nonepileptic events occurred. Utility of video-EEG monitoring allows to differentiate and classify seizures and epileptic syndromes in childhood, especially absence seizures and psychogenic pseudoepileptic seizures. Its use makes the introduction of appropriate treatment possible.