Regulation of inflammatory processes in ALS: SOCS, activated STAT, and c/EBP ß J.M.H. Anneser , C. Chahli , S. Franz , G.D. Borasio , S. Lorenzl 
Department of Neurology, University of Munich, Munich, Germany
Interdisciplinary Center for Palliative Medicine, University of Munich, Munich, Germany
Elevations of cytokines in ALS indicate that inflammation may contribute to motoneuron (MN) death. However, little is known about the regulation of proinflammatory proteins in ALS.
We used microarray and immunochemical techniques to investigate the role of proteins involved in the regulation of inflammation. We found expressional changes of STAT, suppressor of cytokine signalling (SOCS) proteins, and CCAAT/enhancer-binding protein ß (c/EBP ß).
SOCS proteins are induced on cytokine stimulation via STAT activation and block further cytokine signalling in a negative feedback loop. C/EBP ß is a transcription factor that belongs to the basic leucine zippers.
In SOD-transgenic mice, we observed an induction of SOCS 1 and 3, as well as activated STAT 1 and 3 in glial cells. In MN, SOCS 1 and 3 were down-regulated even in presymptomatic mice, while activated STAT 1 and 3 was unchanged. Furthermore, we found an up-regulation of c/EBP ß isolated from the nuclear fraction of SOD-transgenic animals.
Since the transcriptional targets of c/EBP ß include IL-6, COX 2, and CD 14, activation of c/EBP ß may be a key step in the inflammatory cascade.
The high levels of activated STAT proteins in SOD-transgenic mice demonstrate the involvement of the JAK/STAT pathway in ALS. Accordingly, enhanced SOCS expression may be an attempt to limit the inflammation. Interestingly, SOCS expression is reduced in MN. This may reflect the loss of an important endogenous negative feedback-loop in these cells.
Dr. J.M.H. Anneser
University of Munich, Neurology
Cysteine donor intake is able to reduce rest and exercise-related oxidative stress in Amyotrophic Lateral Sclerosis CC Carlesi , PS Piazza , DA DelCorona , FM Franzini , PA Pompella , MM Mancuso , PA Paolicchi , SG Siciliano 
Neurology, Pisa, Italy
Experimental Pathology, Pisa, Italy
Amyotrophic Lateral Sclerosis (ALS) is a neurological disorder characterized by a progressive upper and lower motor neurons loss. ALS causes are unknown, but accumulating evidences, indicate that oxidative stress is involved in the pathogenesis of this disease. Objective of our work has been to analyze the occurrence of oxidative stress in ALS before and after 3 months of treatment with a cysteine donor food-integrator. We assessed blood levels of the following oxidative stress markers in 16 ALS patients, before and after 45 days of treatment with a food-integrator cysteine donor (Prother, AFR, 10 mg/die): glutathione (GSH), the advanced oxidation protein products (AOPP) and the ferric reducing ability (FRAP). AOPP and FRAP were also measured, besides resting condition, during an incremental hand-grip dynamometer muscle exercise test, performed in the two experimental conditions.
We found that, in resting condition and before therapy, compared to 7 matched controls, FRAP was unchanged, while GSH was decreased (p <0.01) and AOPP increased (p < 0.05) in ALS patients. After therapy a significant decrease (p <0.02), compared to pre-treatment condition, of exercise-related blood AOPP levels in ALS patients occurred, still significant (p<0.004) at three month follow-up. Our results confirm the role of exercise related oxidative stress in sporadic ALS pathogenesis and underline the beneficial effects of antioxidant cysteine-donor therapy to modify blood levels of oxidative stress.
MD CC Carlesi
via Roma 67
Phone: +39 050993046
BIA in patients with amyotrophic lateral sclerosis - a prospective study in 54 patients S Endruhn , P Kühnlein , AC Ludolph , AD Sperfeld 
Department of Neurology, University of Ulm, Ulm, Germany
Pharmacological treatment of patients suffering from amyotrophic lateral sclerosis (ALS) is reduced on riluzole, which has shown to heighten life expectancy. The main objective of therapy is represented by symptomatic strategies. The nutritional status is of interest as there is evidence that patients with ALS underlie altered energy metabolisms.
The bioelectrical impedance analysis (BIA) is an established method to evaluate the composition of body compartments. The scope of this study was to analyse several BIA parameters in a longitudinal follow-up design, which reflect information on malnutrition in the course of ALS.
54 consecutive patients with possible or definite ALS underwent BIA analysis at study inclusion, after 3, 6 and 9 months. At each time-point all patients further received clinical investigations, measurements of body weight and performance of the revised ALS-FRS.
There was a decrease in body weight in male patients and body mass index (BMI) in all patients. The values of the ALS-FRS were in a continuous decline from baseline to month 9. BIA revealed a decrease amongst the basic metabolic rate (GU), the body cell mass (BCM) and the phase angle (PA). There were significant correlations both of PA and ALS-FRS and GU and ALS-FRS.
BIA is a stable and reliable method for the objective analysis of the nutritional status of ALS patients and might be a useful tool for a well-timed initiation of supportive feeding protocols in order to preserve functional capabilities.
Polymorphism in the Cu/Zn superoxide dismutase (SOD1) gene in a large ALS family A Felbecker , P Steinbach , S Winter , J Kassubek , AC Ludolph , A-D Sperfeld 
University of Ulm, Ulm, Germany
Background: Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are well-known causes of amyotrophic lateral sclerosis (ALS) and account for approximately 20% of familial ALS cases. While reduced penetrance of SOD-associated ALS is not unusual, there has been no description of a gene polymorphism of a SOD1 mutation in one family. Methods: We describe a family with 13 members that suffered from ALS and another 61 healthy relatives. All affected members of the family that were alive, could be contacted and gave informed consent for genetic testing were examined. DNA of those individuals and some not affected family members was extracted and tested for mutations in the SOD1 gene. Results: The clinical presentation of all affected individuals was comparable with slow progression and predominant signs of the second motoneuron. Out of 5 tested affected individuals 2 had the E100K mutation of the SOD1 gene, whereas the 3 remaining did not have a SOD1 mutation. Out of 5 healthy family members, one individual carried the mutation. Discussion: We could not find any correlation between the E100K mutation and clinical parameters. We conclude that the SOD1 mutation is not the main pathogenetic factor in this family to cause ALS. Our results contribute to the ongoing discussion concerning the relevance of SOD1 mutations in the pathogenesis of ALS and support the hypothesis of a heterogeneous cause of the disease, where the SOD1 mutation is only one of many pathogenetic factors.
University of Ulm, Neurology
Oberer Eselsberg 45
Angiogenin (ANG) mutations are associated to sporadic amyotrophic lateral sclerosis (sALS) in the German population Rubén Fernández-Santiago, MSc,1 Sabine Hoenig, MD,2 Peter Lichtner, PhD,3 Anne-Dorte Sperfeld, MD,2 Thomas Illig, PhD,4 Johanna Anneser, MD,5 Christoph Münch, MD,2,6 Stephan Zierz, MD,7 Thomas Gasser, MD,1 and Albert Ludolph, MD2
1 Department for Neurodegenerative Disorders, Hertie-Institute for Clinical Brain Research, Eberhard-Karls University, Tuebingen, Germany
2 Department of Neurology, University Hospital of Ulm, Ulm, Germany
3 Institute for Human Genetics, GSF-National Research Centre for Environment and Health, Neuherberg, Germany
4 Institute for Epidemiology, GSF-National Research Centre for Environment and Health, Neuherberg, Germany
5 Department of Neurology, Grosshadern University Hospital, Ludwig-Maximilians University, Munich, Germany
6 Department of Neurology, Charité Humbolt-University Hospital Berlin, Berlin, Germany
7 Department of Neurology, University Hospital Halle-Wittenberg, Halle, Germany
Mutations in the angiogenin (ANG) gene were recently found to be associated with familial and sporadic ALS selectively in the Scottish/Irish population. To investigate whether ANG modulates ALS susceptibility in the German population, we performed mutational screening in a large cohort of 581 German ALS patients and 365 neurological controls by sequencing the entire coding region of the ANG gene.
We identified four heterozygous pathogenic missense mutations located in the signal peptide (F(-13)L) and mature ANG protein (K17I, I46V and K54E) of four sporadic ALS male patients (0.7%). Each of the mutations may lead to inactivation/ mislocalization of ANG. Mutations F(-13)L and K54E have not been previously associated with ALS. Clinically, ANG-associated ALS was characterized in our sample by a predominant lower motor neuron manifestation of disease and a reduced average life-time expectancy (34 months).
Our results suggest that mutations in the ANG gene may be a cause of increased susceptibility to ALS in the Caucasian population. ANG is a downstream effector of vascular endothelial growth factor (VEGF), another cytokine upregulated by hypoxia linked to ALS. VEGF has been shown to be a modifier of human and mouse ALS exerting neuroprotective effects in SOD1G93A mice model. We hypothesize that, similar to VEGF, or even as part of the same pathway, ANG may play a neuroprotective role by promoting neural survival in motor neurons.
Mr R Fernandez-Santiago
Hertie-Institute for Clinical Brain Research, Neurodegenerative Disorders
Lack of association of VEGF gene polymorphisms with sporadic amyotrophic lateral sclerosis (sALS) in a Polish population Golenia A , Tomik B , Slowik A , Zawislak D , Ostrowska M , Szczudlik A 
Department of Neurology, Jagiellonian University, Medical College, Krakow, Poland
Introduction. Genetic factors involved in the pathogenesis of sALS still remain unknown. Vascular endothelial growth factor (VEGF) is one of the candidate genes. VEGF is a cytokine that has a protective function via angiogenic, neurotrophic, gliotrophic and anti-apoptotic activity. It has become evident that VEGF play a role in sALS pathogenesis. Transgenic mice expressing reduced VEGF levels develop late-onset motor neuron degeneration, reminiscent of ALS. The recent studies showed that -634G/C and -2578C/A polymorphisms of VEGF gene were associated with a greater risk of sALS.
The aim of the study. To check a possible association between -634G/C and -2578C/A polymorphisms of VEGF gene and a risk of sALS as compared to healthy controls in a Polish population.
Materials and Methods. We have included 204 unrelated patients with sALS and 348 unrelated healthy controls matched for age and sex. The definite or probable diagnosis of ALS was established according to El Escorial Criteria. The polymorphisms were studied by polymerase chain reaction and restricted enzyme digestion.
Results. VEGF -634G/C and VEGF -2578C/A genotype frequency in patients with sALS and controls were very similar and did not influence the risk of occurrence of sALS (p=0.57; p=0.81).
Conclusion. We found that -634G/C and -2578C/A polymorphisms of VEGF gene are not connected with the greater risk of sALS in Polish population. Our results are in line with data from German, Dutch and London populations.
MD AG Golenia
Department of Neurology
3 Botaniczna Street
Keywords: ALS, Polymorphisms, VEGF
Prior presentation: no
Potential conflict of interest: no
All authors did approve:yes
Permission to print: yes
Presenting author: MD Golenia A
Ubiquitous overexpression of Hsp27 does not attenuate disease in mutant G93A SOD1 mice J Krishnan , K Vannuvel , D Kiraly , W Robberecht , L Van Den Bosch 
Katholieke University Leuven, Leuven, Belgium
The exact pathogenic mechanism of mutant SOD1 induced motor neuron death in ALS is still elusive and is thought to involve both oxidative stress and protein aggregation. These two phenomena are known to induce heat shock proteins (Hsp’s) which protect cells through their chaperoning and anti-apoptotic activity. Loss of Hsp27 from motor neurons preceding disease has been suggested to contribute to their degeneration in ALS mice. Moreover, several Hsp’s, notably Hsp27, have been shown to be protective in several in vitro ALS models. However, we recently showed a lack of protective effect of Hsp27 against mutant SOD1-dependent cell death in N2a cells. In this study, we show corroborative results using the ALS mice. Mice that overexpress the human G93A SOD1 mutant were crossed with ubiquitous Hsp27 overexpressors and the resulting double transgenic mice (Dtg) were tested. The Dtg mice did not live longer, nor had significant delayed onset of disease compared to their G93A SOD1 littermates. No evidence that motor neurons were protected in these mice was found histologically. Also no difference in the activation, nor in the levels of members of the apoptotic machinery was found in these mice. In conclusion, Hsp27 alone does not seem to be sufficient to protect against the devastating effects of mutant SOD1.
Ms J Krishnan
Katholieke University Leuven, Lab of Neurobiology
Herestraat 49, O&N2
PO Box 1022
ALS - how to think of life D. Lulé , T. Matuz , AC. Ludolph , N. Birbaumer , A. Kuebler 
University of Ulm, Neurology, Ulm, Germany
Eberhard-Karls-University of Tübingen, Medical Psychology and Behavioural Neurobiology, Tuebingen, Germany
National Institutes of Health (NIH), NINDS, Human Cortical Physiology, Bethesda, United States
Demands to facilitate end-of-life decisions for ALS patients become increasingly strong. Our research over the last years aimed to explore the circumstances of life with ALS.
In five studies of our research group (I-V) we investigated social, emotional and cognitive factors in ALS patients.
Our studies indicate that there is no correlation of physical disabilities and depression or Quality of life (QoL) in ALS (I). Depression rate correlated negatively with education and depended on the patient’s attitude. A high QoL was possible at any stage of the disease and QoL key values were adjust to the circumstances. In several studies, cognitive deficits have been confirmed for some ALS patients. In our study (II), for some severely restricted ALS patients a positive correlation of physical restrictions and verbal and non-verbal memory was confirmed which might suggest a shift of attention. ALS patients presented a low aggression rate and a high degree of self control (III). Furthermore, they presented a changed emotional processing as they evaluated social stimuli as more positive and less arousing (IV) and had an impaired ability to recognize negative facial expressions (III). Functional studies revealed a changed cortical pattern for social information processing (V).
Only a minor fraction of patients decide to prolong their lives with e.g. NIV often because of an expected low QoL. Our data confirms that a positive life experience in ALS is possible at any stage of the disease.
Cell death pathways differ in several mouse models with motoneuron disease : gene profiling of pure motoneuron populations F E PERRIN , G BOISSET , A LATHUILIERE , M DOCQUIER , O SCHAAD , P DESCOMBES , A C KATO 
Department of Basic Neuroscience, Faculty of Medicine, Geneva, Switzerland
Genomics Platform, National Center of Competence in Research “Frontiers in Genetics”, Geneva, Switzerland
Institute for Neuroscience of Montpellier, INSERM U583, Montpellier, France
We combined laser capture microdissection with microarray technology to identify candidate genes that may be involved in motoneuron degeneration. We have analyzed three animal models that develop motoneuron degeneration. These include transgenic SOD1G93A mice and two autosomal-recessive mouse mutants that develop spontaneous mutations, pmn and wobbler.
Three major observations were made: first, there was only a small number of genes that were differentially expressed in motoneurons at a pre-symptomatic age in all three mouse models. Secondly, one of the earliest signs of motoneuron death appears to be the up-regulation of the gene coding for the intermediate filament vimentin. We showed that the vimentin protein was expressed in the cytoplasm of the motoneurons of all three animals at a pre-symptomatic age and accumulated during the disease progression. Thirdly, a time-course analysis of the motoneurons at a symptomatic age in transgenic SOD1G93A surprisingly showed a modest de-regulation of only three genes associated with cell death pathways but a massive up-regulation of genes involved in cell growth.
These experiments define early molecular changes that may be involved in the pathogenesis of motoneurons leading to cell death and suggest that one of the earliest signs of motoneuron death may be the appearance of an up-regulation of vimentin. In addition these results demonstrate that there is no widespread induction of cell death associated genes in motoneurons of SOD1G93A mice.
Dr F PERRIN
Institut for Neurosciences of Montpellier, Inserm U583
Evaluation of patients with lower motor neurone disorders K Pasternak , M Ficek , B Tomik , A Szczudlik 
Department of Neurology, Jagiellonian University Medical College, Krakow, Poland
BACKGROUND: It could be difficult to initially distinguish ALS with clinically predominant lower motor neuron (LMN) involvement from alternative diagnoses of LMN disorders. LMN syndromes have been known to positively affect prognosis.
MATERIAL AND METHODS: We retrospectively analysed a group of 68 patients with initially isolated lower motor neurone (LMN) signs at the disease onset selected out of 450 consecutive MND patients who were evaluated in Krakow MND Center of Department of Neurology in 2003-2006.
RESULTS: Forty two patients had flail-arm (FA) or flail-leg (FL) phenotype of ALS ( M:F-3.2:1; age of onset 48.8+/-11.2 years; disease duration 42.9+/-35 months), 15 had Kennedy’s syndrome (SBMA; 15 men; age of onset 43.3+/-11.5 years; disease duration 15.3+/-6.2 years), 6 had spinal muscular atrophy (SMA) (6 men; age of onset 11.4+/-6.2 years; disease duration 26.4+/-6.6 years) and 5 had post-polio syndrome (PPS; 3 men, 2 women; age of onset 47.6+/-9.4 years; disease duration 10+/-2.3 years). FA and FL presented with isolated LMN signs for a longer period of time before they finally developed ALS but had a longer life span. First LMN presentation in each of diseases were: proximal weakness of the upper limbs in FA, unilateral foot drop in FL, distal weakness of the lower limbs in SBMA and distal paresis of the lower limbs in post-polio syndrome.
CONCLUSION: Isolated involvement of LMN signs at presentation was diagnosed as different types of motor neuron diseases.
Institute of Neurology, Department of Neurology Jagiellonian University Medical College
Paraoxonase gene polymorphisms and susceptibility to sporadic ALS in Italian population: preliminary results S Penco , C Ricci , L Cozzi , G Greco , R Causarano , MC Patrosso , A Marocchi , F Giannini , S Battistini 
Medical Genetics, Niguarda Hospital, Milan, Italy
Neurology, University of Siena, Siena, Italy
Background: Among "susceptibility" genes potentially contributing to the development of ALS, attention has been focused recently on Paraoxonase (PON1 and PON2) genes involved in detoxification of organophosphorous compounds. One study reported a significant association of the R allele at Q192 in PON1 and the C allele at S311 in PON2 with SALS in a Polish population. Subsequent studies found no association between these polymorphisms and SALS. However an association was found for some PON1 promoter polymorphisms.
Objectives. To investigate the possible association between PON1 (L55M and Q192R), and PON2 (C311S) and the risk of sporadic ALS as compared to healthy controls in an Italian population.
Material and Methods : We included 200 SOD1 negative patients with definite or probably SALS diagnosed according to El Escorial Criteria, and 300 controls. All individuals gave their informed consent and were genotyped for the three gene variations.
Results: 100 SALS patients and 200 controls were analyzed: genotype distributions did not significantly differ between patients and controls.
Conclusions: Preliminary results did not found an association between PON1 and PON2 coding polymorphisms with sporadic ALS in this group of Italian patients however a screening on the remain samples is ongoing. Furthermore analysis of the PON1 promoter region is currently in progress in order to determine possible role of polymorphisms affecting protein expression in susceptibility to SALS.
Dr S Penco
Niguarda Ca' Granda Hospital, Medical Genetics
Piazza Ospedale Maggiore 3
firstname.lastname@example.org, Phone: +390264442803
Brain endophenotype of extremity and bulbar onset ALS in diffusion tensor imaging T Peschel , J Kaufmann , R Heil , N Bodammer , R Dengler , HJ Heinze , J Grosskreutz 
University of Sheffield, Sheffield, United Kingdom
Medical School Hannover, Hannover, Germany
Otto-von-Guericke-University, Magdeburg, Germany
In ALS, recent MRI voxel based analyses have characterized the disease process early in vivo. Widespread cortical atrophy was found extending from the sensorimotor cortex to the dorsolateralprefrontal cortex into frontal cortical areas. White matter abormalities were found along the corticospinal tract (CST) and in frontal projections in diffusion imaging. It is as yet unclear whether extremity or bulbar onset ALS display different brain endophenotypes.
We examined 17 mildly affected (ALSFRS ~40) ALS patients and 17 age-matched controls in a GE 1.5 T scanner. Diffusion tensor image sets were generated from 12 planes with eddy current correction. The apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were analyzed in group comparisons and correlated to clinical parameters using SPM software.
All ALS patients showed a significant reduction in FA along the corticospinal tract (CST) compared to controls. ADC images sets, however, were more sensitive in detecting these changes, and showed abnormal diffusion in the white matter extending into frontal regions. In extremity onset ALS only frontal white matter ADC abnormalities were found, whereas in bulbar ALS, the CST and adjacent areas were affected.
This in-vivo neuropatholoy MRI study sheds light on the distribution pattern of diffusion abnormality of different onset type ALS. It raises again the question whether these subtypes are caused by specific degenerative processes.
Feasibility of multicenter analyses of voxel-based morphometry in Amyotrophic Lateral Sclerosis T Peschel , J Kassubek , J Kaufmann , L Emmerich , A Mangold , R Dengler , H Becker , J Grosskreutz 
University of Sheffield, Sheffield, United Kingdom
In ALS, voxel-based morphometry (VBM) has detected cortical atrophy to a variable degree. It is unclear whether these differences are due to variability in the disease process or patient groups, small sample size or differences in technical procedures. We aimed to demonstrate the feasibility of multicenter VBM analyses in ALS patients from three centers.
53 ALS patients and 53 controls were scanned in different 1.5 T scanners to produce high resolution T1 brain volume images (Hannover n=21/GE, Ulm n=15/Siemens, Magdeburg n=17/GE). Images were analyzed with the optimized VBM protocol in SPM2 software. The study template was created from all controls. Modulated grey matter data were analyzed. Group comparisons were based on the general linear model by ANOVA with the imaging center as nuisance variable. Global normalization was perfomed by proportional scaling.
The precentral gyrus and premotor areas displayed marked atrophy in the multicenter analysis, whereas frontal atrophy was not detected. We found inconsistent extent of frontal cortical atrophy in within center analyses.
This to our knowlegde largest VBM analysis in ALS patients demonstrated that atrophy of the primary motor cortex is the dominant common pathology in ALS. The multicenter approach provides new perspectives for the use of VBM in clinical studies.
Further report on stable motor improvement after prolonged beta-lactam antibiotic therapy in ALS PS Piazza , CC Carlesi , FM Falorni , VL Volpi , SG Siciliano 
Neurology, Pisa, Italy
Neurology, Pisa, Italy
Neurology, Pisa, Italy
Neurology, Pisa, Italy
Neurology, Pisa, Italy
Intractability of amyotrophic lateral sclerosis (ALS) is mainly to the unknowledge of aetiology of motor neuron degeneration. Glutamate-mediated neurotoxicity was suggested as a mechanism of motor neuron death, and astroglial glutamate transporter GLT1, the transporter responsible for the majority of total glutamate transport in the central nervous system (CNS), as a possible therapeutic target.
In this report we present a 60-yr-old age male with one year history of ALS, undergone to tracheotomy and percoutaneous endoscopic gastrostomy some months before, wheelchair bound since six months, who, after a cyclic therapy with antibiotics, basically beta-lactams, for several months, showed a significant improvement of skeletal and respiratory muscle function detected with spirometry. Besides their bactericide properties, beta-lactam antibiotics, including penicillin and ceftriaxone, have been considered putative therapeutic agents for ALS, by a modulation of astroglial GLT1 glutamate transporter expression. This observation, in supporting a likely role of glutamate-induced neurotoxicity for ALS, confirms occasional previous reports of a possible therapeutic effect of beta-lactam antibiotics in ALS, also in advanced stage of this disease.
MD PS Piazza
via Roma, 67
Electromyographic approach to ALS patients in Italy: a retrospective study L Testa , R Tarletti , GD Oggioni , N Nasuelli , L Mazzini , R Cantello 
Department of Neurology, “Eastern Piedmont “University, “Maggiore della Carità” Hospital, Novara, Italy
The aim of our study was to evaluate the electromyographic approach to Amyotrophic Lateral Sclerosis (ALS) in Italy. Among patients attending a third level ALS centre we revised 71 electromyographic examinations (EMG) performed as part of the diagnostic work-up in 41 different laboratories. We evaluated the EMG according to the methodological suggestions proposed by the Italian Society of Clinical Neurophysiology (SINC). SINC criteria require needle examination of at least 2 muscles in both cervical and lumbar regions and 1 muscle in bulbar region. Just 28% fulfilled these criteria; 83% were complete for limbs and 37% had a cranial muscle study. Motor and sensory conduction study requires the examination of at least 3 different limbs searching for conduction blocks and studying F waves. Motor conduction study was performed in 90% but in only 68% patients it included both upper and lower limbs. Conduction blocks were searched in 58% and F wave in 37% patients. Sensitive nerves were explored in 79% but just in 41% both in upper and lower limbs. In conclusion, needle examination of limbs muscles was sufficiently performed but cranial muscles were poorly studied and paraspinal muscles were neglected. Nerve conduction studies were insufficiently considered to exclude nerve conduction blocks neuropathy. Hence EMG at the time of diagnosis in Italy is often incomplete to support the El Escorial criteria. International guidelines are needed to uniform EMG diagnosis in ALS.
doctor G D oggioni
maggiore della carità Hospital, neurology
V.LE MAZZINI 18
Concurrence of amyotrophic lateral sclerosis (ALS) and autoimmune diseases (AID) in an inbred Italian family. L Testa , L Corrado , G.D. Oggioni , S Cozzi , N Nasuelli , L Mazzini , S D'Alfonso 
Dept of Neurology “ Maggiore della Carità” Hospital, novara, Italy
Dpt of Medical Sciences, University of Eastern Piedmont, novara, Italy
We report the case of a 45-years-old woman with Systemic Lupus Erythematosus (SLE) who developed seven years later, a progressive bulbar and motor palsy typical of ALS. Intravenous cyclophosphamide and immunoglobulin and plasmapheresis therapies were uneffective. At present the patient has a severe tetraparesis, the speech is not understandable and feeding tube was necessary for nutrition. This clinical history suggests that the proband has two distinct diseases (SLE and ALS) and not an ALS-mimic syndrome. She is negative for SOD1 mutations. The patient belongs to an inbred family from a small Sardinian village with a history for both ALS and autoimmune diseases. Her parents are consanguineous. Her maternal uncle developed at age 17 a severe form of rheumatoid arthritis (RA) and the first signs of MND at age 58. He died at 60 with an ALS diagnosis. A maternal third degree cousin presented autoimmune polyendocrinopathy type III and ALS at age 45. Several other relatives have AID with no present sign of MND: two sisters (52 and 44 years) and two distant cousins (60 and 55 years) with SLE and a maternal uncle (76 years) with RA. Although the individual analyses indicate that the proband has two distinct diseases (SLE and ALS) and not an ALS-mimic syndrome, the concurrence of ALS and AID in three patients of this family might suggests a possible common pathogenetic mechanism.
doctor G D oggioni
maggiore della carità Hospital, neurology
V.LE MAZZINI 18
A first-pass genome-wide scan for age of onset to amyotrophic lateral sclerosis using 2,336 microsatellite markers
V B Tripathi , A Al-Chalabi C Simpson , V Hansen , J Knight , B Neale , P Sham , C Shaw , N Leigh , J Powell ,
MRC Centre for Neurodegenration Research, London, United Kingdom
MRC SGDP Centre, London, United Kingdom
ALS is a progressive fatal neurodegenerative disorder of motor neurons. In about 2% of all cases, mutations are found in SOD1 gene. Several candidate gene screens have yielded inconsistent results, possibly because of phenotypic heterogeneity. We performed a genome-wide scan for association with age of onset using microsatellite markers in DNA pools using a two-stage design. Individuals from a clinic-based population were selected on the basis of disease severity. Controls were from the same geographical region and matched for age and sex. DNA pools were made with the highest and lowest quintiles for age of onset, and the remainder split by clinical phenotype. Microsatellites were selected to target gene-dense regions or candidate genes and typed on an ABI 3100 genotyper. For the first pass, results were prioritised using metaregression of allele frequencies against phenotype weighted by the inverse of measurement variance. There were 300 cases and 300 controls with 60 in each of the age of onset pools, 123 in limb onset and 57 in bulbar onset pool. Results were available for 2336 microsatellite markers. Metaregression analysis was neither statistically conservative nor liberal. In the top 1% of results, two markers were in the same small region of chromosome 3, and another was in a locus associated with age of onset of Alzheimer disease on chromosome 10. This method allows putative age of onset genes to be identified for follow-up studies.
Dr. V B Tripathi
Institute of Psychiatry, Department of Clinical Neurosciences
PALS with PEG: nutritional issues M.C. Tuccio , P. Bongioanni , K. Nardi , F. Fulceri , M. Cagnacci , G. Pepe , M.R. Metelli , M. Azadegan , B. Rossi 
Neurorehabilitation, Neuroscience Dpt, University of Pisa, Pisa, Italy
Laboratory of Clinical Biochemistry, Experimental Pathology Dpt, University of Pisa, Pisa, Italy
Nutritional Service, University of Pisa, Pisa, Italy
Patients suffering from Amyotrophic Lateral Sclerosis(PALS) have to face with progressive nutritional problems leading at last to percutaneous endoscopic gastrostomy(PEG) placement.
We evaluated 9 spinal-onset(s-o) or bulbar-onset(b-o) PALS (mean age ¬± SD: 62¬±10 yrs) with PEG. We collected many nutritional data (weight, body mass index, blood prealbumin, albumin, transferrin and lymphocyte count, body cell mass index(BCMI), and other soft tissue analysis parameters) before and 2 months (t2) after PEG insertion. In a 6-PALS subgroup (mean age ¬± SD: 62¬±7 yrs), we studied nutritional data also after 8 months (t8) from PEG insertion.
Comparing the 9 PALS before PEG insertion and at t2, we found BCMI mean values significantly (p<0,05) reduced at t2 (4.6¬±1.4vs5.5¬±2.5), while prealbumin (27.0¬±4.8vs25.4¬±7.3 mg/dl) and albumin (4.5¬±0.3vs4.4¬±0.4 g/dl) increased, and transferrin and lymphocyte counts remained unchanged.
Moreover, considering a subgroup of patients evaluated at t2 and t8, we found reduced BCMI mean values overtime (5.6¬±2.6, 4.4¬±1.4, and 4.2¬±1.6, before PEG insertion and at t2 and t8, respectively) together with increased prealbumin (26.5¬±8.8vs27.2¬±6.2vs29.0¬±5.3 mg/dl), albumin (4.2¬±0.3vs 4.5¬±0.3vs4.6¬±0.5 g/dl) and transferrin (224.7¬±35.9vs245.2¬±31.7vs258.7¬±34.4 mg/dl) mean values, whereas lymphocyte counts remained unchanged.
Our results support the concept that PEG placement, although disease still keeps progressing, can improve PALS nutritional status.
Cardiovascular autonomic responses to orthostatic stress in patients with „Flail-arm“ and „Flail-leg“ phenotypes of amyotrophic lateral sclerosis M Tutaj , K Pasternak , M Ficek , A Kyrcz , B Tomik , A Szczudlik 
Department of Neurology, Jagiellonian University Medical College, Krakow, Poland
Background: Flail-arm (FA) and flail-leg (FL) are ALS phenotypes with distinct clinical course that are sometimes associated with better survival. Cardiovascular autonomic system (CAS) disturbances can negatively affect prognosis in many diseases and have been described in ALS. However, there are no data on CAS function in FA or FL.
The aim of the study was to assess cardiovascular responses to orthostatic stress in patients with limb- (ALS-L) and bulbar-onset (ALS-B) forms of classical ALS and in patients with FA/FL phenotypes.
Methods: In 6 ALS patients with FA (2) or FL (4) phenotypes and 8 with classical ALS: ALS-L (5), ALS-B (3), we continuously monitored ECG, respiration and blood pressure (BP) during supine rest and head-up tilt and assessed low (LF) and high frequency (HF) spectral powers of RR intervals (RRI).
Results: Head-up tilt induced similar RRI responses in all groups. However, LF and HF powers of RRI during tilt were reduced in ALS-B. Furthermore, significant orthostatic falls in systolic, diastolic and mean BP were observed in ALS-B and in those FA/FL patients who had developed signs of trunk involvement as the second manifestation of ALS, but not in ALS-L patients or FA/FL patients who had had involvement of a different limb following initial FA/FL signs.
Conclusion: Sympathetic vascular but not cardiac responses to orthostatic stress are impaired in the subgroup of FA/FL patients with trunk involvement as the second manifestation of their disease.
Dr M Tutaj
Jagiellonian University Medical College, Neurology
Mutant SOD1 induces a motor axonopathy in the zebrafish embryo A Van Hoecke , R Lemmens , L Vandenbosch , P Carmeliet , W Robberecht 
KULeuven, Leuven, Belgium
KULeuven, Leuven, Belgium
The development of small animal models is of major interest to unravel the etiology, pathogenesis and treatment of neurodegenerative diseases. We have investigated the zebrafish Danio rerio as a potential model to study one such neurodegenerative disease, amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons. Mutations in superoxide dismutase (SOD1) been identified in familial ALS. Overexpression of mutant human SOD1 in zebrafish embryos induced an axonopathy characterized by abnormal outgrowth of motor axons specifically. The deleterious effect was dose dependent and observed for all mutations tested. To demonstrate the usefulness of this model as a screening tool for chemical or genetic modifiers, we investigated whether this mutant SOD1-induced axonopathy could be influenced by factors known to modify mutant SOD1-associated motor neuron degeneration: expression of vascular endothelial growth factor (VEGF) and wildtype SOD1. Induction of VEGF in the zebrafish rescued the motor axon defect, while lowering of VEGF resulted in a more severe phenotype. Similarly, co-expression of wild type SOD1, known to aggravate disease in rodent models of ALS, exacerbated the axonal defects. This novel zebrafish model for ALS will permit large scale genetic and chemical screening and thus may facilitate the identification of new therapeutic targets in motor neuron degeneration.
Effect of radiotherapy versus botulinum toxin A injection into the salivary glands in Amyotrophic Lateral Sclerosis; a comparative study. J.G. Weikamp , D.A.X. Schinagl , C.C.P. Verstappen , B.J.M. de Swart , H.J. Schelhaas , M.J. Zwarts 
Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands
Background: Drooling is a common and invalidating symptom in patients with Amyotrophic Lateral Sclerosis (ALS). Radiotherapy and botulinum toxin (BTX) injections are now well accepted as alternative treatment options to the more traditional and often disappointing drug management of this symptom. Still, a well-designed prospective and controlled trial to compare these two methods has never been performed. The purpose of this study is to answer the question which treatment is the most effective one with the least side effects.
Methods: In a prospective, randomized, single blind, controlled trial we will evaluate efficacy and tolerability of ultrasound-guided BTX-A injections in parotids and/ or submandibular glands in comparion with radiotherapy. Radiotherapy is performed by administering a single dosage of 7.0Gy bilateral to the parotids glands and the posterior parts of the submandibular glands. The effect of treatment will be compared between baseline and four weeks after the intervention by various questionnaires, the number of used paper handkerchiefs during two days and quality of live measurements like the Amyotrophic Lateral Sclerosis Assessment Questionnaire (ALSAQ-40). Side effects will also be monitored by quantitative measurement of bulbar function.
Results: Inclusion started summer of 2006 and five patients have now been treated. The first results will be presented at the meeting.
MA J.G. Weikamp
Radboud University Nijmegen Medical Centre, speech and language pathology
6500 HB Nijmegen
Disease progression and its predictors in Amyotrophic Lateral Sclerosis K. Kollewe, S. Petri, K. Krampfl, U. Mauss, R. Dengler, B. Mohammadi
Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive, degenerative disorder of upper and lower motor neurons with a progressive bulbar or limbic muscular atrophy. The average survival in ALS patients lies between 3 to 5 years, but the survival in an ALS-patient is known to have a wide variety and is considered to be difficult to predict.
Methods: We identified 479 patients in our ALS Database who fulfilled the diagnostic criteria for probable or definite ALS according to the El Escorial criteria of the World Federation of Neurology and who have regularly been followed at our ALS-clinic in a time period from 1996 to 2006. Demographic, clinical and electro-physiological data were collected at each clinical visit. The ALSFRS-R (ALS Functional Rating Scale Revised) score is performed at each follow-up visit at approximately 3-month intervals for evaluations of disease progression. Major interventions such as non-invasive ventilation (NIV) or percutaneous endoscopic gastrostomy (PEG) are recorded during follow-up. The descriptive analyses and correlation (Pearson test two sided) were performed with SPSS-Software.
Results: The study cohort consisted of 479 patients, 269 men and 210 women. The mean age at the time of disease onset was 58 years (SD 12), ranging from 25 to 89 years. 165 of the analyzed patients had died at time of analyse. Survival from symptom-onset ranged from 4 months up to 11.9 years. 5% of patients died within 1 year after symptom-onset (age: 43-77 years), 81% (age: 34-85 years) of the patients lived one to five years after first symptoms, 12% (Age: 38-75 years) survived five to 10 years, and 2% lived longer than 10 years after the first symptom.
Discussion: Older age at onset, bulbar onset, lower initial FVC, shorter time between first symptom and first visit, lower ALSFRS-R score at first contact, higher rate of ALSFRS-R score between first symptom and first contact or during whole disease correlate with a higher risk of death. Predictors of survival are important for designing new trials and to randomize patients correctly.
Dr. med. K. Kollewe
Carl-Neuberg-Str.2C II/ App.511
Tele: 0511-16 05 52 2
Mobil: 0179-97 94 78 4
Guidelines for the preclinical studies on SOD1 mutant mice: report from the 142nd ENMC international workshop. C. BENDOTTI1 , A. LUDOLPH2
1.Mario Negri Institute for Pharmacological Research, Milan , Italy
2.University of Ulm, Germany
Co-author: The ENMC group for the establishment of guidelines for the conduct of preclinical and proof of concept studies in ALS/MND models
A matter of debate in the field of ALS is the overall failure to translate the efficacy of treatment strategies developed in animal models into clinical trials. The choice of therapeutic agents to be tested in human ALS has been based mainly on the efficacy of these drugs in one of the most commonly used model of ALS, the transgenic mouse carrying several copies of the human SOD1 transgene mutated in G93A. However, as pointed out in a recent systematic review, most of therapeutic studies conducted in SOD1 mice suffer of limited methodological quality which lead to a lack of reproducibility and consistency ( Benatar, 2007). Hence , the necessity of establish standard methods for the design and the interpretation of drug testing studies involving SOD1 mutant mice. During a workshop held in Holland last year, a group of clinical and pre-clinical researchers, with the support of the European Neuromuscular Centre (ENMC), developed guidelines for the community to improve the quality of the studies. Recommendations concerning the characteristics of SOD1 mutant mice, the beginning of treatment, design of experiments,the outcome measures, statistical evaluation of data and publication politic will be discussed.
Benatar M.(2007). Lost in translation: Treatment trials in the SOD1 mouse and in human ALS. Neurobiol Dis. 26:1-13.
Istituto Mario Negri,
Via Eritrea 62
Tel: +39 02 39014317
Fax: +39 02 3546277 +39 02 39001918
Fluorescence-based clinical proteomics of human peripheral blood mononuclear cells from ALS patients Giovanni Nardo1,2, Silvia Garbelli3, Stefania Mantovani3, Mario Salmona2, Gabriele Mora3,Caterina Bendotti2 and Valentina Bonetto1,2 1Proteomic Unit, Dulbecco Telethon Institute
2Mario Negri Institute for Pharmalogical Research, Milan
3Fondazione Salvatore Maugeri, IRCCS, Pavia
Diagnosis of amyotrophic lateral sclerosis (ALS) is achieved late in the disease, when 30-50% of motor neurons have already been lost. Moreover, survival rates vary among individual patients and can be from a few months to years from diagnosis. Identification of specific biomarkers would be of great importance for early and accurate diagnosis, better treatment of the disease and for following response to therapy in clinical trials. Advanced proteomic technologies offer promising approaches to identify panels of protein biomarkers of disease. In particular, fluorescence-based difference gel electrophoresis (DIGE) technology provides a powerful quantitative component to proteomic experiments involving two-dimensional (2D) gel electrophoresis. DIGE allows the detection of subtle changes in protein abundance with statistical confidence while controlling for gel-to-gel variation. We investigated the feasibility of using peripheral blood mononuclear cells (PBMC) for biomarker discovery. We performed a pilot study with PBMC from healthy individuals and ALS patients grouped on the basis of the ALS functional rating scale (ALSFRS-R) in >24 and ≤24. By using DIGE-technology we found that there is a selective group of proteins with differences in abundance in the various samples. We are now carrying out mass spectrometry analysis to identify all the differentially expressed proteins. Furthermore, we identified tyrosine-nitrated proteins under physiological and pathological conditions and compared their level of oxidation using fluorescence-based 2D Western blotting. The three major nitrated proteins detected, actin, ATP synthase and CD41 have a higher level of nitration in the ALS patients than in controls. The identification and quantification of the other nitrated proteins are in progress. In conclusion, PBMC analysis by fluorescence-based proteomic technologies seems to be a promising approach to monitor changes associated to the disease.