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We describe considerations and strategies for developing a nuclear magnetic resonance (NMR) sample preparation method to extract low molecular weight metabolites from high-salt spent media in a model coculture system of phytoplankton and marine bacteria. Phytoplankton perform half the carbon fixation and oxygen generation on Earth. A substantial fraction of fixed carbon becomes part of a metabolite pool of small molecules known as dissolved organic matter (DOM), which are taken up by marine bacteria proximate to phytoplankton. There is an urgent need to elucidate these metabolic exchanges due to widespread anthropogenic transformations on the chemical, phenotypic, and species composition of seawater. These changes are increasing water temperature and the amount of CO

Phytoplankton-derived metabolites fuel a large fraction of heterotrophic bacterial production in the global ocean, yet methodological challenges have limited our understanding of the organic molecules transferred between these microbial groups. In an experimental bloom study consisting of three heterotrophic marine bacteria growing together with the diatom Thalassiosira pseudonana, we concurrently measured diatom endometabolites (i.e., potential exometabolite supply) by nuclear magnetic resonance (NMR) spectroscopy and bacterial gene expression (i.e., potential exometabolite uptake) by metatranscriptomic sequencing. Twenty-two diatom endometabolites were annotated, with nine increasing in internal concentration in the late stage of the bloom, eight decreasing, and five showing no variation through the bloom progression. Some metabolite changes could be linked to shifts in diatom gene expression, as well as to shifts in bacterial community composition and their expression of substrate uptake and catabolism genes. Yet an overall low match indicated that endometabolome concentration was not a good predictor of exometabolite availability, and that complex physiological and ecological interactions underlie metabolite exchange. Six diatom endometabolites accumulated to higher concentrations in the bacterial co-cultures compared to axenic cultures, suggesting a bacterial influence on rates of synthesis or release of glutamate, arginine, leucine, 2,3-dihydroxypropane-1-sulfonate, glucose, and glycerol-3-phosphate. Better understanding of phytoplankton metabolite production, release, and transfer to assembled bacterial communities is key to untangling this nearly invisible yet pivotal step in ocean carbon cycling.

International audience; Background and purposeThe aim was to determine the genetic background of unknown muscular dystrophy in five French families.MethodsTwelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested invitro.ResultsFive patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation.ConclusionsThe mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families.

Background The efficacy of enzyme replacement therapy (ERT) in patients at an advanced stage of Pompe disease has only been addressed in a few studies. Our objective was to assess the long term effects of ERT in a cohort of patients with severe Pompe disease. Methods We identified patients from the French Pompe Registry with severe respiratory failure and permanent wheelchair use (assisted walk for a few meters was allowed) when starting ERT. Patients' medical records were collected and reviewed and respiratory and motor functions, before ERT initiation and upon last evaluation were compared. Results Twelve patients (7 males) were identified. Median age at symptom onset was 24 years [IQR = 15.5; 36.0]. At baseline ventilation was invasive in 11 patients and noninvasive in one, with a median ventilation time of 24 h [IQR = 21.88; 24.00] (min 20; max 24). ERT was initiated at a median age of 52.5 years [IQR = 35.75; 66.50]. Median treatment duration was 55 months [IQR = 39.5; 81.0]. During observational period no adverse reaction to ERT was recorded, five patients (41.67%) died, three decreased their ventilation time by 30, 60 and 90 min and two increased their assisted walking distance, by 80 and 20 m. Conclusion Some patients at a very advanced stage of Pompe disease may show a mild benefit from ERT, in terms of increased time of autonomous ventilation and of enlarged distance in assisted walk. ERT can be initiated in these patients in order to retain their current level of independence and ability to perform daily life activities.

Objectives The aim of the study was to compare prospectively indicator-condition (IC)-guided testing versus testing of those with non-indicator conditions (NICs) in four primary care centres (PCCs) in Barcelona, Spain. Methods From October 2009 to February 2011, patients aged from 18 to 65 years old who attended a PCC for a new herpes zoster infection, seborrhoeic eczema, mononucleosis syndrome or leucopenia/thrombopenia were included in the IC group, and one in every 10 randomly selected patients consulting for other reasons were included in the NIC group. A proportion of patients in each group were offered an HIV test; those who agreed to be tested were given a rapid finger-stick HIV test (€6 per test). Epidemiological and clinical data were collected and analysed. Results During the study period, 775 patients attended with one of the four selected ICs, while 66 043 patients presented with an NIC. HIV screening was offered to 89 patients with ICs (offer rate 11.5%), of whom 85 agreed to and completed testing (94.4 and 100% acceptance and completion rates, respectively). In the NIC group, an HIV test was offered to 344 persons (offer rate 5.2%), of whom 313 accepted (90.9%) and 304 completed (97.1%) testing. HIV tests were positive in four persons [prevalence 4.7%; 95% confidence interval (CI) 1.3–11.6%] in the IC group and in one person in the NIC group (prevalence 0.3%; 95% CI 0.01–1.82%; P

Resume Les formes autosomiques recessives de la maladie de Charcot-Marie-Tooth (AR-CMT) sont caracterisees par un debut infantile et un phenotype severe comparativement aux formes dominantes. Les maladies de CMT reliees aux mutations du gene de la periaxine (PRX) sont rares et sont caracterisees par une demyelinisation majeure restreinte aux nerfs peripheriques. A la suite de la decouverte d’une grande frequence d’une mutation particuliere de la periaxine a l’Ile de La Reunion (E1085fsX4 homozygote), nous avons examine tous les patients francais connus comme porteurs de mutation du gene de la periaxine , soit 24 dont 18 issus de l’Ile de La Reunion (6 familles et 10 cas sporadiques). Les six autres patients sont originaires de la Metropole et sont issus de deux familles comportant deux sujets atteints et deux autres cas sporadiques. On denombre 17 femmes et sept hommes. La marche est acquise avec retard, en moyenne a 3,4 ± 1,6 ans. Un patient n’a jamais acquis la marche. L’index CMTNS (Charcot Marie Tooth Neuropathy Score) est en moyenne a 24,5 ± 8,1. Sept patients ont perdu la marche en moyenne vers 24 ± 22 ans. D’autres symptomes sont notes : une scoliose majeure presente au dela de 12 ans, parfois compliquee d’un syndrome respiratoire restrictif, une deformation des pieds, d’un strabisme, d’un glaucome et d’une myopie. Lorsque les vitesses de conduction sont enregistrables, il existe un ralentissement majeur de la vitesse de conduction motrice du nerf median ( periaxine doit etre envisagee chez les patients presentant une neuropathie demyelinisante severe de debut infantile associee a une scoliose. Le pronostic fonctionnel est pejoratif etant donne que 29 % des patients sont en fauteuil roulant et presentent une insuffisance respiratoire restrictive. Ainsi, le conseil genetique est recommande au sein de ces familles consanguines.

Background: Multiple mitochondrial DNA (mtDNA) deletions usually have a mendelian inheritance secondary to mutation in nuclear genes. One of these is the Twinkle gene whose mutation is responsible for autosomal dominant progressive external ophthalmoplegia (PEO). The number of reported cases with mainly myopathic symptoms and possible nervous system involvement related to Twinkle gene mutation is limited. We present a new French family of whom two members displayed myopathy and neuropathy associated with PEO, and we perform a clinical review in light of other observations reported in the literature. Methods: The proband, one son and the daughter have been investigated. Southern blot analysis and long-range PCR assay have been performed from muscle biopsy specimens. Coding exons and flanking intron regions of polymerase gamma (POLG) and DNA helicase (Twinkle) genes were sequenced. Results: Multiple mitochondrial DNA deletions have been found and sequencing of the Twinkle gene showed the change p.R374Q. Conclusion: Two other families from the literature also had the R374Q mutation. Symptoms reported in association with this mutation were myopathy, peripheral neuropathy, dysarthria and/or dysphagia, respiratory insufficiency and parkinsonism. Respiratory insufficiency caused by chest wall weakness was reported in other families with different Twinkle gene mutations, and one might provide exercise intolerance, dysarthria and/or dysphagia as symptoms in favor of the diagnosis. Occurrence of impressive emaciation was a peculiarity in our family.

Revue Neurologique - In Press.Proof corrected by the author Available online since samedi 25 avril 2015

Resume La myopathie facio-scapulohumerale est une affection autosomique dominante dont le locus a ete identifie dans la region telomerique du chromosome 4 en 4q3S. Elle parait avoir un taux de mutation elevee. Observation Une enfant presente des l'enfance les signes d'une forme severe de myopathie facio-scapulohumerale en l'absence d'antecedents familiaux. Le diagnostic ne sera confirme qu'a l'âge de 23 ans grâce a l'etude moleculaire retrouvant la deletion. Celle-ci est retrouvee avec une faible intensite chez le pere, qui apparait comme porteur d'une mutation sous forme d'une mosaique germinale sans expression clinique. Conclusion Cette observation illustre la possibilite de forme severe de la myopathie facio-scapulohumerale et le caractere dominant de l'affection que la clinique simple ne permet pas de reconnaitre. Elle souligne donc l'importance de l'enquete en biologie moleculaire et les difficultes du conseil genetique.

The aim of the study was to compare prospectively indicator-condition (IC)-guided testing versus testing of those with non-indicator conditions (NICs) in four primary care centres (PCCs) in Barcelona, Spain.From October 2009 to February 2011, patients aged from 18 to 65 years old who attended a PCC for a new herpes zoster infection, seborrhoeic eczema, mononucleosis syndrome or leucopenia/thrombopenia were included in the IC group, and one in every 10 randomly selected patients consulting for other reasons were included in the NIC group. A proportion of patients in each group were offered an HIV test; those who agreed to be tested were given a rapid finger-stick HIV test (€6 per test). Epidemiological and clinical data were collected and analysed.During the study period, 775 patients attended with one of the four selected ICs, while 66,043 patients presented with an NIC. HIV screening was offered to 89 patients with ICs (offer rate 11.5%), of whom 85 agreed to and completed testing (94.4 and 100% acceptance and completion rates, respectively). In the NIC group, an HIV test was offered to 344 persons (offer rate 5.2%), of whom 313 accepted (90.9%) and 304 completed (97.1%) testing. HIV tests were positive in four persons [prevalence 4.7%; 95% confidence interval (CI) 1.3-11.6%] in the IC group and in one person in the NIC group (prevalence 0.3%; 95% CI 0.01-1.82%; P0.009). If every eligible person had taken an HIV test, we would have spent €4650 in the IC group and €396,258 in the NIC group, and an estimated 36 (95% CI 25-49) and 198 persons (95% CI 171-227), respectively, would have been diagnosed with HIV infection. The estimated cost per new HIV diagnosis would have been €129 (95% CI €107-153) in the IC group and €2001 (95% CI €1913-2088) in the NIC group.Although the number of patients included in the study was small and the results should be treated with caution, IC-guided HIV testing, based on four selected ICs, in PCCs seems to be a more feasible and less expensive strategy to improve diagnosis of HIV infection in Spain than a nontargeted HIV testing strategy.

Autosomal recessive Charcot-Marie-Tooth disease (AR-CMT) is often characterized by onset in early childhood and severe phenotype compared to the dominant forms. CMT disease associated with periaxin gene (PRX) is rare and characterized by demyelination limited to the major peripheral nerves. Following the discovery of a high frequency of a specific periaxin gene mutation (E1085fsX4 homozygote) in the Reunion Island, we examined all French patients known as carriers of the periaxin gene mutation. There were 24 patients. Eighteen were from the Reunion Island (6 families and 10 sporadic cases). The six remaining patients were in two families, each with two affected individuals, and two sporadic cases. The series included 17 female and seven male patients. Walking was acquired late, on average at 3.4±1.6 years. One patient never learned to walk. The Charcot Marie Tooth Neuropathy Score (CMTNS) averaged 24.5±8.1. Seven patients had been wheelchair-bound since the age of 24±22. Other symptoms were: scoliosis most often observed after the age of 12 years and sometimes complicated by a restrictive respiratory syndrome; foot deformity in 24 patients; strabismus; glaucoma; myopia. When conduction recordings are available, median nerve motor conduction was slow (10m/s), associated with a major lengthening of distal latencies. Study of the periaxin gene should be considered in patients with severe demyelinating neuropathy associated with early infantile scoliosis. This disease leads to major disability (29% of patients in this series were wheelchair-bound) and to respiratory insufficiency. Genetic counselling is highly recommended for consanguineous families.

We report a severe phenotype of Charcot-Marie-Tooth (CMT) disease type 1E caused by a novel p.Phe84Leufs*24 PMP22 point mutation. Ultrastructural examination of a nerve biopsy showed non- or partly myelinated axons which were surrounded by "onion bulb" formations mainly composed of concentric basement membranes and characterized by the presence of prominent concentric or longitudinal collagen fibrils interspersed with basement membranes. PMP22 point mutations are rare and responsible for polyneuropathies often demyelinating with onion bulb formations composed of concentric and redundant basement membranes. Entrapment of prominent collagen fibrils within onion bulb formations is unusual, even in the large spectrum of CMT disease with long duration and severe damage.

Setting: Tuberculin testing is an accepted method for screening pregnant women for tuberculosis. The interpretation of tuberculin reactivity in bacillus Calmette-Guerin (BCG)-immunized pregnant women is still in debate. Objective: Four related issues were addressed: (1) The effect of pregnancy on tuberculin reactivity; (2) the effect of age differential on tuberculin reactivity; (3) the effect of repeated immunization with BCG; and (4) the risk of developing tuberculosis during pregnancy or a 3-year post-partum period. Design: We performed tuberculin testing in 840 healthy Chilean women in the 32nd to 34th week of pregnancy; 807 had been immunized with 1 or more doses of BCG. There were 3 age groups: 177 were ≥ 19 years old, 534 were 20–29 years old, and 129 were ≥ 30 years old. All women in the study were followed at least 3 years post-partum. Results: Women ≤ 19 years old and non-pregnant women of similar age studied in the same geographical area had a similar distribution of the size of tuberculin reactions. Over 50% of all BCG-immunized women in each group had tuberculin reactions ≥ 10 mm. A differential effect of different doses of BCG was significant only in 20- to 29-year-old women. None of the women in this study developed tuberculosis during pregnancy or a 3-year post-partum observation period. Conclusions: Healthy, BCG-immunized pregnant women may have positive tuberculin reactions without having an increased risk for tuberculosis. The incidence of tuberculosis and the BCG immunization status need to be considered in the development of policy recommendations for diagnosis and treatment of Mycobacterium tuberculosis infection in pregnant women.

Los proyectos de monitoreo de aves han proporcionado datos valiosos para el desarrollo de indicadores biológicos que evalúan el estado de los hábitats naturales y agrícolas; sin embargo, los avances han sido menores en los ambientes urbanos. En este estudio se utilizaron los datos del monitoreo de aves de dos ciudades climáticamente diferentes (Bruselas y Barcelona; período 2002–2010) para generar dos indicadores urbanos multiespecíficos que valorasen las tendencias temporales en la abundancia del conjunto de las aves urbanas. Para hacer esto, utilizamos dos enfoques conceptuales distintos, uno basado en una lista de especies de amplia distribución en las ciudades europeas (WSEC) y otro basado exclusivamente en especies de amplia distribución a nivel de ciudad (WSC), independientemente de las aves de otras ciudades. Los dos indicadores dieron un patrón general similar, aunque un 3% de diferencia entre ellos en cuanto a los valores de cambio promedio anual se encontró en ambas ciudades. Esto sugiere que los valores producidos por los indicadores urbanos pueden diferir dependiendo de la aproximación conceptual y, por tanto, por la lista de especies utilizada para generarlos. Ambos indicadores pueden tener su propio interés y pueden ser tratados como complementarios., Bird monitoring projects have provided valuable data for developing biological indicators to evaluate the state of natural and agricultural habitats. However, fewer advances have been made in urban environments. In this study we used bird monitoring data from 2002 to 2012 in two cities with different climates (Brussels and Barcelona), to generate two multi–species urban indicators to evaluate temporal trends on abundance of urban avifauna. To do this we used two different conceptual approaches, one based on a list of widespread species in European cities (WSEC) and another based exclusively on species widespread at city level (WCS) regardless of the birds occurring in other cities. The two indicators gave a similar general pattern, although we found a 3% difference in the mean annual change in both cities, thus suggesting that the values provided by urban indicators may differ depending on the conceptual approach and, hence, by the species list used to generate them. However, both indicators may have their own value and could be treated as complementary indices.

The proportion of tuberculin reactors in a population and the intensity of tuberculin reactions have been shown to increase with increasing exposure to mycobacterial infection, eg, repeated BCG immunization. These observations suggested that tuberculin reactivity would become uniformly high in individuals with a high mycobacterial load who did not have a known cause of anergy. Since tuberculin reactivity has been measured to evaluate the possible genetic regulation of responses to mycobacteria in humans, it is important to study its behavior under conditions of ongoing, maximal exposure to mycobacteria. In the present study, we determined the mean size of tuberculin reactivity in BCG-immunized and unimmunized patients with pulmonary tuberculosis of recent onset, and the stability of tuberculin reactions during and after treatment of pulmonary tuberculosis.Serial tuberculin testing was performed on patients with newly diagnosed active pulmonary tuberculosis diagnosed over a period of 2 years at the National Institute for Respiratory Diseases in Santiago, Chile. The first tuberculin test was performed at the time of diagnosis in 58 patients. Repeated tuberculin testing was performed 2 weeks later in 15 patients with initial reaction sizes15 mm. Four additional tuberculin tests were performed, one each at 3-months intervals in 42 patients regardless of the size of the initial tuberculin reaction.Tuberculin reactions at entry had a unimodal distribution in patients both with and without BCG scars (14.8 +/- 5.0 mm and 16.5 +/- 5.2 mm, respectively). A second tuberculin test in patients with initial reaction sizes15 mm showed a moderate, statistically significant increase in the mean reaction size (PPD1: 10.1 +/- 3.2 mm; PPD2: 11.9 +/- 4.8 mm). Repeated tuberculin testing over 1 year revealed no significant changes in reaction size. The mean reaction sizes were 15.8 +/- 5.0 mm at entry, 15.5 +/- 5.4 mm at 3 months, 17.2 +/- 5.2 mm at 6 months, 17.0 +/- 5.1 mm at 9 months, and 16.7 +/- 54 mm at 12 months. The standard deviation of a random observation within patients was 5.3 mm. The largest variations due to increased reactivity after 6 months of treatment were observed in patients with reaction15 mm at entry compared with hyperergic patients, and in BCG-immunized patients compared to unimmunized patients.In the presence of an ongoing mycobacterial infection, patients without anergizing conditions express a tuberculin reactivity that is relatively constant during and after treatment of pulmonary tuberculosis. The size and stability of the reactions seem to be determined by individual conditions that include the tuberculin reactivity at the time of diagnosis and the BCG immunization status.

Multiple mitochondrial DNA (mtDNA) deletions usually have a mendelian inheritance secondary to mutation in nuclear genes. One of these is the Twinkle gene whose mutation is responsible for autosomal dominant progressive external ophthalmoplegia (PEO). The number of reported cases with mainly myopathic symptoms and possible nervous system involvement related to Twinkle gene mutation is limited. We present a new French family of whom two members displayed myopathy and neuropathy associated with PEO, and we perform a clinical review in light of other observations reported in the literature.The proband, one son and the daughter have been investigated. Southern blot analysis and long-range PCR assay have been performed from muscle biopsy specimens. Coding exons and flanking intron regions of polymerase gamma (POLG) and DNA helicase (Twinkle) genes were sequenced.Multiple mitochondrial DNA deletions have been found and sequencing of the Twinkle gene showed the change p.R374Q.Two other families from the literature also had the R374Q mutation. Symptoms reported in association with this mutation were myopathy, peripheral neuropathy, dysarthria and/or dysphagia, respiratory insufficiency and parkinsonism. Respiratory insufficiency caused by chest wall weakness was reported in other families with different Twinkle gene mutations, and one might provide exercise intolerance, dysarthria and/or dysphagia as symptoms in favor of the diagnosis. Occurrence of impressive emaciation was a peculiarity in our family.

Sole et al.1 utilized different camera angles to document a reversible left ectropion in a patient with myasthenia gravis before and after a neostigmine. They documented the left ectropion from the camera angle position to the patient's left and above horizontal, which highlighted the reddish lower lid eversion. After the neostigmine, the camera position was moved to the patient's right and below the horizontal level. In addition, the left eye is hypertropic. This new camera angle and repositioning of the …

Limb girdle muscular dystrophies are rare genetic diseases. Despite constant progress in genetics and biochemistry, the pathogenic mechanisms are not completely understood. Calpainopathy (LGMD2A) has been reported to be the most frequent autosomal recessive form of muscular dystrophy in several populations. Point mutations in CAPN3 are difficult to identify and the analysis is long and costly. The use of western blot does not seem to provide the expected sensitivity and specificity.We studied all the patients diagnosed in the neuromuscular center of Bordeaux (France) with confirmed calpainopathy in order to establish the appropriate diagnostic approach (inclusion criteria: muscular biopsy with calpain 3 western blot study, two mutations in CAPN3). Patients with highly suspected calpainopathy (same criteria with only one mutation) were also analyzed.Our 13 patients belonged to 10 different families. Four patients had a normal western blot for calpain (WBn). We found high phenotypic variability with frequent atypical signs. The WBn group had less severe disease (a statistically significant later age of onset, a tendency toward lower CK levels and a slower disease course). We extended this comparison to the single mutation patients and we found the same results.Considering the lack of sensitivity of western blot protein analysis in LGMD2A, a normal western blot for calpain should not halt the genetic analysis. The western blot result seems to have prognostic value. A normal western blot may help genetic testing by highlighting some mutational hot spots in the CAPN3 gene.

The booster or enhancement effect of repeated tuberculin skin testing in Calmette-Guérin bacillus (BCG)-vaccinated young adults was studied in 208 first-year medical, nursing, and medical technology students in Santiago, Chile, where BCG vaccine is usually administered at birth and at 6 and 14 yr of age. Thirty-three students had no BCG scar, 62 had one scar, 71 had two scars, and 42 had three scars. The mean age for each group was 19 yr. All students were healthy and had no known exposure to tuberculosis or history of tuberculosis or other mycobacterioses. The size in millimeters of induration of the first tuberculin reaction (PPD1) was clearly correlated with the number of BCG scars: 2.3 +/- 4.6 for no scars; 6.7 +/- 6.7 for one scar; 10.9 +/- 5.9 for two scars, and 13.2 +/- 5.3 for three scars. The second tuberculin reaction (PPD2), performed 2 wk later on the contralateral forearm, showed a marked increase in reactivity. The increase in reaction size was most evident in students who had BCG scars but who were initially PPD negative (less than 10 mm). Smaller increases were observed in students without BCG scars, and also in those who had BCG scars but who were initially tuberculin positive (greater than or equal to 10 mm). The persistence of the booster effect was evaluated by performing PPD3 1 yr later. PPD1-negative students with BCG scars maintained the increased level of reactivity to PPD2 after 1 yr. An immunizing effect of tuberculin testing was suggested in 11 nonimmunized students who were initially PPD negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

Limb girdle muscular dystrophy type 1b (LGMD1B), due to LMNA gene mutations, is a relatively rare form of LGMD characterized by proximal muscle involvement associated with heart involvement comprising atrio-ventricular conduction blocks and dilated cardiomyopathy. Its clinical and genetic diagnosis is crucial for cardiac management and genetic counselling. Seven LMNA mutations have been previously reported to be responsible for LGMD1B.We describe the neurological and cardiologic features of 14 patients belonging to 8 families in whom we identified 6 different LMNA mutations, 4 of them having never been reported. Results. Eleven patients had an LGMD1B phenotype with scapulohumeral and pelvic-femoral involvement. Thirteen patients had cardiac disease associating conduction defects (12 patients) or arrhythmias (9 patients). Seven patients needed cardiac device (pacemaker or implantable cardiac defibrillator) and two had heart transplantation.This study allowed us to specify the clinical characteristics of this entity and to outline the first phenotype/genotype relations resulting from these observations.

Necrotizing autoimmune myopathy (NAM) is a group of severe acquired myopathies, characterized by prominent myofiber necrosis with little or no muscle inflammation. They can be misdiagnosed as muscular dystrophies, preventing initiation of appropriate therapy. Specific auto-antibody anti-Signal recognition protein is observed in 16% of cases. Recently, auto-antibodies against 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR) were identified in NAM patients by one group, especially in statin exposed patients. Here we report the first European series of patients. We detected and quantified anti-HMGCR auto-antibodies using addressable laser bead immunoassay that we developed. Patients (n = 38) were 44 ± 19 years old (3 pediatric cases) and sex ratio male: female was 0.22. Statin exposure was recorded in 40% of patients. Patients suffered from myalgia (44%), and had a muscular deficit (92%). Subacute onset (less than 6 months) was noted for most of them (n = 18), nevertheless 10 patients had a slow progressive muscular deficit (10 months to years). Severe proximal weakness was observed (72%) and three patients were bedridden at the diagnosis. All patients had increase CK level (6630 ± 5990 UI/L). CK level correlated with muscular strength (r2 = −0.64, p = 0.005) and to anti-HMGR titer (r2 = −0.5, p = 0.04). None patients presented signs of pulmonary involvement. Mean duration of treatments (steroids, immunosupressant and/or IgIV) was 30.1 ± 36.1 [3–42] months, and to date it was not possible to stop the treatment for any patient. This the first study confirming the observation and the description of anti-HMGCR NAM. The majority of patients we detected was statin-naive, had a severe weakness and need prolonged treatments. Anti-HMGCR auto-antibodies testing may be warranted in dystrophic like patients. Anti-HMGR titer correlates with CK level suggesting their role in physiopathogenesis.

Less than half of all subjects display a normal configuration of the Circle of Willis, according to anatomical studies. Variations of the middle cerebral artery (MCA) such as duplication or accessory MCA are infrequent but nevertheless have important clinical implications. We report the case of two patients with these variations of the MCA and their repercussions in the management of acute stroke are discussed.Case 1: a 53 year old male with a 2 hour history of sensory motor syndrome; a transcranial Doppler (TCD) scan revealed asymmetrical speeds in the MCA. Spontaneous perforation of the MCA was suspected and we therefore decided to perform a magnetic resonance angiography scan before administering fibrinolytics. The magnetic resonance angiography scan showed an accessory MCA lying ipsilateral to the lesion. We interpreted the anomalies in blood flow detected in the TCD recording as being secondary to this anatomical variation and not due to reperfusion. Following the magnetic resonance angiography study, the possibility of fibrinolysis was ruled out. The patient recovered the neurological deficit in a matter of hours. Case 2: a 21 year old female with headaches and transient hemiparesis, who was diagnosed as suffering from migraine with aura. Later, following another stroke, it was shown that the previous clinical symptoms had been secondary to intracranial dissection with embolism in the lenticulostriate arteries and ischemic infarction in that territory. A magnetic resonance angiography scan showed duplication of the ipsilateral MCA.The double vascularisation of the hemisphere in cases of anatomical duplication can give rise to strokes with a better progression and prognosis, despite the occlusion of one of the MCA. The presence of anatomical variations of the MCA can lead to mistaken interpretations of the transcranial Doppler scan and may affect decision making as regards the therapy to be employed in patients with acute stroke.

POEMS is an acronym for polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes. POEMS syndrome is also called Crow-Fukase syndrome, chiefly in Japan. The 5 above mentioned features are not always present at the first examination. The minimal criteria to establish the diagnosis are the presence of a demyelinating and axonal polyneuropathy associated with an IgA or IgG monoclonal gammopathy, the light chain being almost always lambda, and at least 2 of the 8 other features: sclerosing plasmocytoma, endocrinopathy, skin changes, organomegaly, Castleman's disease, anasarca, papillary edema or thrombocytosis. Among these features, only cutaneous glomeruloid angioma are specific. Ultrastructural identification of uncompacted myelin lamellae on the peripheral nerve biopsy is also a strong argument in favor of the diagnosis. An associated "osteosclerotic" bone lesion must be carefully searched, because its treatment may improve the other features of the syndrome, especially the neuropathy. Cytokines and the vascular growth endothelial factor might play a role in the pathogenesis of this rare multisystemic disorder.

We report the neuropathological data from a familial case of chorea-acanthocytosis with central and peripheral nervous system involvement. At the age of 34, the patient underwent a peripheral nerve biopsy which was analyzed by light- and electron microscopy. These studies showed a selective reduction in the large diameter myelinated fibre population, with several clusters of regeneration. Remyelinating fibers surrounded by flattened Schwann cell processes were also present. The patient died at the age of 44, and post-mortem macroscopic examination of the brain showed marked atrophy of the caudate. Histological examination of paraffin sections showed almost complete depletion of neurons in the caudate, with severe astrocytic gliosis. The putamen and pallidum were slightly less severely depleted of neurons, but with marked astrocytic gliosis. Diffuse mild gliosis was also evidenced, by immunohistochemistry with anti-GFAP, in the thalamus and subcortical white matter.

Inclusion body myositis (IBM) is a severe form of idiopathic inflammatory myopathy. A predominantly T CD8+ lymphocytic infiltrate, with focally non-necrotizing muscular fiber invasion, and rimmed-vacuoles are specific histological signs. A few cases of IBM associated with other dysimmune diseases have been reported, but only once with systemic sarcoidosis. We report three cases of muscular sarcoidosis associated with IBM. This very uncommon observation suggests that major complex of histocompatibility, soluble factors, cytokines and adhesion molecules could be involved. Our cases are a novel example of associated dysimmune diseases.

We report 13 French patients with proximal myotonic myopathy. PROMM is a recently delineated multisystem disorder with dystrophic myopathy, myotonia and cataracts. This syndrome is genetically distinct from myotonic dystrophy (DM) by the absence of abnormal CTG repeat expansion. The geographical origin varies but 4 families originated from Poland. Of late onset, muscle weakness is diffuse and predominantly affected proximal and axial muscles. Facial involvement and myotonia were moderate or absent, but in all cases myotonic discharges were detected on EMG. 6 patients suffered from myalgia. Cataracts occurred in 11 patients, mainly indistinguishable from those in DM. Cardiac arrythmia occurred in 7 patients. Muscle biopsy revealed rare structural changes of the muscle fibers and selective type I atrophy, common in DM, could not be found on morphometric analysis. PROMM has a distinct clinical spectrum from DM which includes a predominantly proximal muscle weakness, with troubling pain, a more favourable prognosis and a different histopathological pattern.

1. Intradermal or subplantar injection of soluble snake venom phospholipase A2 (PLA2) evoked a brisk inflammatory response, with cutaneous vascular permeability increase and paw oedema. 2. These inflammatory processes are mainly the result of arachidonic acid cascade activation and mast cell degranulation. 3. Copper, iron and zinc have an inhibitory effect on vascular permeability increase and paw oedema induced by PLA2. 4. Copper and iron could have not only a direct effect on PLA2 but on enzymes of arachidonic acid cascade. 5. However, zinc have a moderate antiinflammatory activity. This effect could be the result to inhibit PLA2 induced mast cell degranulation.

About 8% of prion disease cases are familial and a few are due to an octapeptide repeat insertion (OPRI) in the prion protein gene. A suitable neuropathological examination has been performed in 20 cases from 9 families and in 3 isolated cases. The number of OPRI ranges from 4 to 9 multiples of 24 base-pair.Results from routine histopathological preparations and from immunohistochemistry performed after special tissue pretreatment were compared with those of molecular genetic investigation.Eight cases with 4 to 7 multiples of OPRI exhibited characteristic elongated deposits in the cerebellar molecular layer, which were visible on slides prepared with antibodies against the prion protein only. Conversely, 6 cases with 8 or 9 multiples of OPRI presented typical plaques already obvious on routine preparations.These variable modifications in the cerebellar molecular layer deserve to be underlined, in particular the elongated deposits which are characteristic for cases presenting 4 to 7 OPRI.

In 1993, WHO and UNICEF recommended the administration of 0.05 ml doses of BCG, instead of 0.1 ml, to newborns. This recommendation was adopted by the Chilean Ministry of Health, using the Mérieux vaccine. Subsequently, different Health Services detected a high percentage of children without BCG scar at the time of their growth and development control.To assess the efficacy of BCG vaccination in a double blind randomized fashion, using two vaccine types and different doses.Newborns of two public hospitals of Santiago were randomly assigned to receive the Tokio or Mérieux BCG strains in doses of 0.05 or 0.1 ml. Ninety five to 125 days after vaccination, vaccine scar was measured and inmunogenicity was assessed using the tuberculin test.Six hundred newborns (150 in each group) were included in the protocol and results were assessed in 408. The percentage of children with a PPD reaction of 0 mm was 9.3, 3.7, 7.8 and 0% with the Mérieux vaccine in doses of 0.1 ml, Tokio vaccines in doses of 0.1 ml, Mérieux vaccine in doses of 0.05 ml and Tokio vaccine in doses of 0.05 ml, respectively. In the same groups the scar diameters were 6.4 +/- 3.4, 7.3 +/- 2.7, 5.6 +/- 2.8 and 7.3 +/- 2.9 mm. The observed differences for each group are significant, depending on the type of strain and dose, but favoring the Tokio type of vaccine.The BCG scar diameters obtained in this study are similar to those obtained in previous works in 1984 and 1986. This scar is the evidence of vaccination that nurses detect in health controls. Therefore the use of 0.1 doses for vaccination, that result in better scars and PPD response, is recommended.

A 58-year-old woman, with recurrent headaches, exhibited cerebellar alaxic gait, anosmia, deafness and a pyramidal syndrome, with a progressive onset. In cerebrospinal fluid there was erythrocytes and siderophages. MRI on T2-weighted images revealed a marginal hypo-intensity, leading to the diagnostic of superficial siderosis of the central nervous system. None haemorragic lesion was found. The patient was given Trientine. Unfortunately she worsened on later examinations.

Germline mosaicism is now well known to account for recurrence of hereditary human disorders. Facioscapulohumeral muscular dystrophy is an autosomal dominant disorder; its locus has been identified in the telomeric region of chromosome 4 at the q35 band. It appears to have a high rate of mutation.A young girl had presented from childhood signs of a severe form of facioscapulohumeral muscular dystrophy, but with no familial history. The diagnosis was ultimately confirmed at the age of 23 years by molecular studies evidencing the deletion. The same abnormality was sparsely found in the child's father who appeared to harbor the mutation as a germline mosaicism with no clinical expression.This case illustrates the possibility of severe facioscapulohumeral muscular dystrophy and the dominant transmission of the disorder which may be clinically occult. It underlines the importance of molecular biology and the difficulties of genetic counselling.

A 72-old-man with a dramatic weight loss, exhibited a mononeuritis multiplex of subacute onset, and myalgia. There was hypoesthesia and weakness on median and ulnar nerves bilaterally, and weakness on left femoral nerve. Electrodiagnostical studies showed an axonal pattern of neuropathy. There was a considerable hypereosinophilia on blood count without any aetiologia found on exhaustive explorations. The idiopathic hypereosinophilic syndrome with a mononeuritis multiplex was suggested. Histological findings were an acute myelino-axonal degeneration with endoneural oedema, without vasculitis nor eosinophil cell on nerve-biopsy. Dramatic improvement occurred with corticosteroids and immunosuppressive treatment.

In patients with HIV infection, disease due to CMV depends on reactivation of the virus. Such reactivation usually occurs at an advanced stage of the disease, when there is severe immunodepression and the CD4+ leukocyte count is100/ml. CMV infection is seen clinically as three syndromes: 1) encephalitis with or without associated meningitis and/or ventriculitis. 2) polyradiculomyelitis affecting the lumbosacral roots, and 3) multifocal senso-motor neuropathy. Diagnosis depends on showing the virus to be present in the CSF, by detecting the early CMV antigen or by conventional culture. There are marked differences between encephalitis, polyradiculomyelitis and multifocal neuropathy in the rentability of viral culture. Whilst in encephalitis CMV culture is negative in most patients, in polyradiculomyelitis the sensitivity of viral culture may be 50-60% and in multifocal neuropathy 15%. The treatment indicated is with ganciclovir or foscarnet. Results depend on the type of neurological disease, degree of involvement when treatment is started and an history of extracerebral CMV infection previously treated with these drugs.

Fourteen patients with peripheral neuropathy and monoclonal IgG of undetermined significance are reported with a retrospective study of the clinical features, electrophysiologic and sural nerve biopsy findings. There were two groups. Five patients had a relapsing chronic sensorimotor polyneuropathy with clinical (5/5), electrophysiologic (4/5) and pathologic (5/5) features compatible with chronic inflammatory demyelinating polyneuropathies (CIDP). The nine others had a slowly progressive sensory (5/9) (SPNP) or sensorimotor (4/9) (SMPNP) axonal polyneuropathy. Four patients of the first group were treated with intravenous human immunoglobulin (400 mg/kg/day for five days) with significant clinical improvement. The motor conduction velocities and distal latencies of two of these patients improved following treatment, thus matching the clinical improvement. Our results on peripheral nerve biopsies confirm the differentiation of patients with CIDP from those with SMPNP and SPNP. There was no specific immunologic serologic reactivity in any of the cases.

We studied retrospectively the cases of neurological forms of Lyme disease observed in two internal and two neurological departments from 1986 till 1993. Twenty five cases have been collected among 15 men and ten women whose mean age was 61 years. Tick bites were previously noticed in 11 cases. Erythema chronicum migrans (ECM) was mentioned in 16 cases mostly on lower limbs. The mean time between ECM and the onset of neurological symptoms was less than 1 month in 11 cases, 2 months in three cases, and 6 months in two cases. Neurological abnormalities were often associated in the same patient. Hyperalgic radiculitis (n = 16), mainly noticed in the ECM territory (n = 10) was only sensitive in six cases and associated with motor deficit in ten. Atypical polyradiculoneuritis was achieved in six cases. Clinical (n = 5) or biological (n = 22) meningitis could occur: CSF was clear with pleiocytosis (132 per mm3), mainly lymphocytic, and hyperproteinorachia (1.2 g/l) with normoglycorachia. An increase of the CSF immunoglobulins G with oligoclonal fragmentation was noticed in 11 cases. Cranial neuropathy was frequent: VII (n = 8), VI (n = 2), III, IV, VIII (n = 1). Encephalitis (with white matter demyelination) resolved partially in two cases. Diagnosis was always confirmed by Borrelia burgdorferi serology (indirect immunofluorescence) with a significant increase of the antibodies titer (n = 17) or a CSF titer1/4 (n = 11). Syphilitic serology was always negative. All patients were treated with parenteral beta lactamins and four with corticosteroids. Outcome was favorable in 20 patients with incomplete resolution of neurological symptoms in two patients.