Living near waterbodies as a proxy of cyanobacteria exposure and risk of amyotrophic lateral sclerosis: a population based case-control study.

BACKGROUND: Epidemiological studies highlighted the possibility that exposure to cyanotoxins leads to the development of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). METHODS: We devised a population-based case-control study in two Italian populations. We used residential proximity of the residence to water bodies as a measure of possible exposure to cyanotoxins. RESULTS: Based on 703 newly-diagnosed ALS cases and 2737 controls, we calculated an ALS odds ratio (OR) of 1.41 (95% CI: 0.72-2.74) for current residence in the vicinity of water bodies, and a slightly lower estimate for historical residence (OR: 1.31; 95% CI: 0.57-2.99). Subjects <65 years and people living in the Northern Italy province of Modena had higher ORs, especially when historical residence was considered. CONCLUSIONS: Overall, despite some risk of bias due to exposure misclassification and unmeasured confounding, our results appear to support the hypothesis that cyanotoxin exposure may increase ALS risk.

Non-length-dependent somatosensory small fiber pathology presenting with restless legs syndrome in pre-motor Parkinson's disease. Evidence from skin biopsy in four patients.

BACKGROUND: The determinants of restless legs syndrome (RLS) occurring in co-morbid association with Parkinson's disease (PD) are currently unknown. METHODS: We performed a skin biopsy in proximal and distal sites of lower limbs in four PD patients, in which RLS had emerged in the pre-motor phase. RESULTS: A reduced somato-sensory intraepidermal nerve fiber (IENF) density mainly in the proximal sites, indicative of non-length-dependent small fiber pathology (SFP), was found in all patients, in absence of electroneurographic signs of large fiber neuropathy. DISCUSSION: The lack of known secondary causes of SFP is consistent with a process intrinsic to PD and, likewise, the absence of known disease conditions associated to RLS, would support the view of a link between the latter disorder and the distal axonopathy. The non-length-dependent pattern of SFP suggest an involvement of the somato-sensory dorsal root ganglia small neurons, consistent with a somato-sensory neuronopathy, which characterizes the RLS in these patients. CONCLUSION: If these findings will be confirmed in a larger cohort of patients, the RLS co-morbid with PD should be regarded as an heterogeneous condition, since the one emerging in the pre-motor phase might represent a prodromal feature of the neurodegenerative disease as an epiphenomenon of somato-sensory SFP. In contrast, for the RLS developing in clinically manifest PD, a possible association with the impairment of the DAergic diencephalo-spinal pathway and the induction by chronic DAergic treatment has been hypothesized.

A Rare Case of Systemic AL Amyloidosis with Muscle Involvement: A Misleading Diagnosis.

Muscle involvement in AL amyloidosis is a rare condition, and the diagnosis of amyloid myopathy is often delayed and underdiagnosed. Amyloid myopathy may be the initial manifestation and may precede the diagnosis of systemic AL amyloidosis. Here, we report the case of a 73-year-old man who was referred to our center for a monoclonal gammopathy of undetermined significance (MGUS) diagnosed since 1999. He reported a progressive weakness of proximal muscles of the legs with onset six months previously. Muscle biopsy showed mild histopathology featuring alterations of nonspecific type with a mixed myopathic and neurogenic involvement, and the diagnostic turning point was the demonstration of characteristic green birefringence under cross-polarized light following Congo red staining of perimysial vessels. Transmission electron microscopy (TEM) confirmed amyloid fibrils around perimysial vessels associated with collagen fibrils. A stepwise approach to diagnosis and staging of this disorder is critical and involves confirmation of amyloid deposition, identification of the fibril type, assessment of underlying amyloidogenic disorder, and evaluation of the extent and severity of amyloidotic organ involvement.

Pesticide exposure assessed through agricultural crop proximity and risk of amyotrophic lateral sclerosis.

BACKGROUND: Epidemiologic studies have raised the possibility that some pesticide compounds induce the neurodegenerative disease amyotrophic lateral sclerosis (ALS), though the available evidence is not entirely consistent. METHODS: We conducted a population-based case-control study in two Italian populations to assess the extent to which residence in the vicinity of agricultural crops associated with the application of neurotoxic pesticides is a risk factor for ALS, using crop acreage in proximity to the residence as an index of exposure. RESULTS: Based on 703 cases and 2737 controls, we computed an ALS odds ratio of 0.92 (95% confidence interval 0.78-1.09) for those in proximity to agricultural land. Results were not substantially different when using alternative exposure categories or when analyzing specific crop types, with the exception of a higher risk related to exposure to citrus orchards and olive groves in Southern Italy, though based on few exposed subjects (N = 89 and 8, respectively). There was little evidence of any dose-response relation between crop proximity and ALS risk, and using long-term residence instead of current residence did not substantially change our estimates. CONCLUSIONS: Though our index of exposure is indirect and subject to considerable misclassification, our results offer little support for the hypothesis that neurotoxic pesticide exposure increases ALS risk.

Magnetic fields exposure from high-voltage power lines and risk of amyotrophic lateral sclerosis in two Italian populations.

The aetiology of amyotrophic lateral sclerosis (ALS), a rare and extremely severe neurodegenerative disease, has been associated with magnetic fields exposure. However, evidence for such a relation in the general population is weak, although the previous null results might also be due to exposure misclassification, or a relationship might exist only for selected subgroups. To test such a hypothesis we carried out a population-based case-control study in two Northern and Southern Italy regions, including 703 ALS cases newly diagnosed from 1998 to 2011 and 2737 controls randomly selected from the residents in the study provinces. Overall, we found that a residence near high-voltage power lines, within the corridors yielding a magnetic fields of ≥0.1 µT, was not associated with an excess disease risk, nor did we identify a dose-response relationship after splitting the exposed corridor according to the 0.1, 0.2 and 0.4 µT cut-points of exposure. These results were confirmed taking into account age at onset, period of diagnosis, sex, geographical area, and length of exposure. Overall, despite the residual possibility of unmeasured confounding or small susceptible subgroups not identified in our study, these results appear to confirm that the exposure to magnetic fields from power lines occurring in the general population is not associated with increased ALS risk.

Heterogeneity in ALSFRS-R decline and survival: a population-based study in Italy.

Very few studies examined trend over time of the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) and factors influencing it; previous studies, then, included only patients attending tertiary ALS Centres. We studied ALSFRS-R decline, factors influencing this trend and survival in a population-based setting. From 2009 onwards, a prospective registry records all incident ALS cases among residents in Emilia Romagna (population: 4.4 million). For each patient, demographic and clinical details (including ALSFRS-R) are collected by caring physicians at each follow-up. Analysis was performed on 402 incident cases (1279 ALSFRS-R assessments). The average decline of the ALSFRS-R was 0.60 points/month during the first year after diagnosis and 0.34 points/month in the second year. ALSFRS-R decline was heterogeneous among subgroups. Repeated measures mixed model showed that ALSFRS-R score decline was influenced by age at onset (p < 0.01), phenotype (p = 0.01), body mass index (BMI) (p < 0.01), progression rate at diagnosis (ΔFS) (p < 0.01), El Escorial Criteria-Revised (p < 0.01), and FVC% at diagnosis (p < 0.01). Among these factors, at multivariate analysis, only age, site of onset and ΔFS independently influenced survival. In this first population-based study on ALSFRS-R trend, we confirm that ALSFRS-R decline is not homogeneous among ALS patients and during the disease. Factors influencing ALSFRS-R decline may not match with those affecting survival. These disease modifiers should be taken into consideration for trials design and in clinical practice during discussions with patients on prognosis.

Erythropoietin in amyotrophic lateral sclerosis: a multicentre, randomised, double blind, placebo controlled, phase III study.

OBJECTIVE: To assess the efficacy of recombinant human erythropoietin (rhEPO) in amyotrophic lateral sclerosis (ALS). METHODS: Patients with probable laboratory-supported, probable or definite ALS were enrolled by 25 Italian centres and randomly assigned (1:1) to receive intravenous rhEPO 40,000 IU or placebo fortnightly as add-on treatment to riluzole 100 mg daily for 12 months. The primary composite outcome was survival, tracheotomy or >23 h non-invasive ventilation (NIV). Secondary outcomes were ALSFRS-R, slow vital capacity (sVC) and quality of life (ALSAQ-40) decline. Tolerability was evaluated analysing adverse events (AEs) causing withdrawal. The randomisation sequence was computer-generated by blocks, stratified by centre, disease severity (ALSFRS-R cut-off score of 33) and onset (spinal or bulbar). The main outcome analysis was performed in all randomised patients and by intention-to-treat for the entire population and patients stratified by severity and onset. The study is registered, EudraCT 2009-016066-91. RESULTS: We randomly assigned 208 patients, of whom 5 (1 rhEPO and 4 placebo) withdrew consent and 3 (placebo) became ineligible (retinal thrombosis, respiratory insufficiency, SOD1 mutation) before receiving treatment; 103 receiving rhEPO and 97 placebo were eligible for analysis. At 12 months, the annualised rate of death (rhEPO 0.11, 95% CI 0.06 to 0.20; placebo: 0.08, CI 0.04 to 0.17), tracheotomy or >23 h NIV (rhEPO 0.16, CI 0.10 to 0.27; placebo 0.18, CI 0.11 to 0.30) did not differ between groups, also after stratification by onset and ALSFRS-R at baseline. Withdrawal due to AE was 16.5% in rhEPO and 8.3% in placebo. No differences were found for secondary outcomes. CONCLUSIONS: RhEPO 40,000 IU fortnightly did not change the course of ALS.

Epidemiology of amyotrophic lateral sclerosis in Emilia Romagna Region (Italy): A population based study.

Our objective was to describe incidence and clinical features of ALS from a prospective population-based study in Emilia Romagna Region (ERR). From 2009 onwards, a prospective registry recorded all incident cases of ALS among residents in the ERR (population, 4.4 million inhabitants), involving 17 neurological departments. For each patient, detailed demographic and clinical information was collected by caring physicians. Results showed that from 1 January 2009 to 31 December 2011, 347 patients received a new diagnosis of ALS with a crude incidence rate of 2.63/100,000/year. There was micro-geographic heterogeneity throughout ERR, with higher incidence rates in the low density population (3.27/100,000) (p < 0.01). ALS patients have been more frequently employed in agriculture than the general ERR population (8.64% vs. 4.6%, p < 0.01). Clinical features were similar to those described in previous population based studies. In conclusion, we report incidence rates similar to those reported by European registries, reflecting good accuracy of our prospective study. We confirmed previous studies reporting higher incidence rates in rural areas and among agricultural workers. Although genetics has been gaining increasing importance in ALS aetiology, some epidemiological data are still unexplained. Identifying geographical areas or populations with high incidence rates can be a starting point for identifying environmental risk factors. Further studies having this specific aim can shed light on these topics.

Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a fatal, untreatable prion encephalopathy. Previous studies showed that doxycycline is effective in in-vitro and in-vivo models of disease, and patients with CJD who received compassionate treatment with doxycycline showed increased survival time compared with historical series. We therefore did a randomised, double-blind study of doxycycline versus placebo in CJD. METHODS: We recruited patients older than 18 years old who had a diagnosis of definite or probable sporadic CJD or genetic forms of the disease via Italian reference centres and the French national referral system. Patients were randomly assigned (ratio 1:1) to receive oral doxycycline (100 mg daily) or placebo under double-blind conditions from the day of randomisation to death. Centralised randomisation was done independently of enrolment or evaluation of patients using a minimisation method in Italy and a simple randomisation in France. Participants, caregivers, and clinicians were masked to group assignment. The primary efficacy variable was the survival time from randomisation. Interim analyses were planned to detect a significant effect of treatment as early as possible. This trial is registered with EudraCT, 2006-001858-27 for the Italian study and 2007-005553-34 for the French study. FINDINGS: From April 12, 2007, to Aug 19, 2010, in Italy, and from Jan 30, 2009, to Jan 10, 2012, in France, 121 patients with CJD were enrolled in the study, 62 of whom were randomly assigned to the treatment group and 59 to the placebo group. The first interim analysis showed absence of superiority of doxycycline compared with placebo, and the trial was stopped for futility. Efficacy analyses did not show significant differences between patients treated with doxycycline and placebo with regard to survival times (HR 1.1, 95% CI 0.8-1.7, p=0.50). Serious adverse events were judged not to be related to treatment, whereas a relation was deemed probable or possible for five non-serious adverse events that occurred in each treatment group. INTERPRETATION: Doxycycline at a dose of 100 mg per day was well tolerated but did not significantly affect the course of CJD, at variance with the results of previous observational studies. Our experience could be useful in the design of large multinational controlled trials of potential anti-prion molecules in this rare disease. FUNDING: Agenzia Italiana Farmaco, Italian Ministry of Health, AIEnP, and French Ministry of Health.

Multi-target action of the novel anti-Alzheimer compound CHF5074: in vivo study of long term treatment in Tg2576 mice.

BACKGROUND: Alzheimer disease is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The pathological hallmarks includes extracellular amyloid plaques and intraneuronal neurofibrillary tangles, but the primary cause is only partially understood. Thus, there is growing interest in developing agents that might target multiple mechanisms leading to neuronal degeneration. CHF5074 is a nonsteroidal anti-inflammatory derivative that has been shown to behave as a γ-secretase modulator in vitro and to inhibit plaque deposition and to reverse memory deficit in vivo in transgenic mouse models of Alzheimer's disease (AD). In the present study, the effects of a long-term (13-month) treatment with CHF5074 on indicators of brain functionality and neurodegeneration in transgenic AD mice (Tg2576) have been assessed and compared with those induced by a prototypical γ-secretase inhibitor (DAPT). RESULTS: To this end, plaque-free, 6-month-old Tg2576 mice and wild-type littermates were fed with a diet containing CHF5074 (125 and 375 ppm/day), DAPT (375 ppm/day) or vehicle for 13 months. The measured indicators included object recognition memory, amyloid burden, brain oligomeric and plasma Aß levels, intraneuronal Aß, dendritic spine density/morphology, neuronal cyclin A positivity and activated microglia. Tg2576 mice fed with standard diet displayed an impairment of recognition memory. This deficit was completely reverted by the higher dose of CHF5074, while no effects were observed in DAPT-treated mice. Similarly, amyloid plaque burden, microglia activation and aberrant cell cycle events were significantly affected by CHF5074, but not DAPT, treatment. Both CHF5074 and DAPT reduced intraneuronal Aß content, also increasing Aß40 and Aß42 plasma levels. CONCLUSIONS: This comparative analysis revealed a profoundly diverse range of clinically relevant effects differentiating the multifunctional anti-inflammatory derivative CHF5074 from the γ-secretase inhibitor DAPT and highlighted unique mechanisms and potential targets that may be crucial for neuroprotection in mouse models of AD.

Exposure to pesticides and risk of amyotrophic lateral sclerosis: a population-based case-control study.

A few epidemiologic studies have suggested an association of agricultural work and pesticides exposure with a severe degenerative disease of the motor neurons, amyotrophic lateral sclerosis (ALS), though conflicting results have also been provided. We investigated through a population-based case-control study the possible relation between overall occupational exposure to pesticides and ALS risk in the northern Italy municipality of Reggio Emilia. By administering a questionnaire, we investigated occupational history and leisure-time habits of the 41 ALS patients diagnosed in the 1995-2006 period, and of 82 age- and sex-matched randomly sampled population controls. More cases than controls were found to have been exposed to pesticides for at least six months (31.7% vs 13.4%, respectively), in all cases within the occupational environment. In a conditional logistic regression model, we found an excess ALS risk associated with exposure to pesticides, with a relative risk of 3.6 (95% confidence interval 1.2-10.5). Such association persisted after inclusion in the statistical analysis of potential confounders. Despite the limited statistical stability of the risk estimates, these results appear to indicate that occupational exposure to pesticides is a risk factor for ALS, suggesting the need to further investigate this issue.

CHF5074, a novel gamma-secretase modulator, restores hippocampal neurogenesis potential and reverses contextual memory deficit in a transgenic mouse model of Alzheimer's disease.

The effects of compounds interfering with gamma-secretase, the enzymatic complex responsible of the formation of the amyloid-beta (Abeta) peptide from amyloid-beta protein precursor (AbetaPP), on plaque deposition in transgenic mouse models of Alzheimer's disease are known but scanty data are available on the effects of these drugs on brain plasticity. We evaluated the effects of long-term treatment with CHF5074, a new gamma-secretase modulator, on hippocampal neurogenesis, cortical synaptophysin levels, and contextual memory in transgenic mice carrying the double Swedish mutation of AbetaPP (Tg2576). Six-month old Tg2576 mice were treated with CHF5074 (375 ppm in the diet) up to 15 months of age. Age-matched control transgenic and wild-type mice received standard diet. Compared to wild-type animals, transgenic controls showed a significant decrease in the number of doublecortin-positive neuroblasts in dentate gyrus, synaptophysin intensity in the cortex, freezing to context in the contextual fear conditioning test. Compared to transgenic controls, CHF5074 treatment of Tg2576 mice resulted in a significant attenuation of the neurogenesis impairment in hippocampus (p=0.036), normalization of synaptophysin levels in cortex (p< 0.001), attenuation of plaque burden in the cortex (p=0.033), increases astroglial reaction around plaques (p=0.001), and attenuation of activated microglia (p=0.040). These effects were associated to a complete reversal of contextual memory deficit (p=0.006). Contextual memory significantly correlated with synaptophysin immunoreactivity in the cortex (r=0.548, p=0.0038).

Central pontine and extrapontine myelinolysis despite careful correction of hyponatremia: clinical and neuropathological findings of a case.

We report clinical, radiological and pathological findings in a patient with central pontine and extrapontine myelinolysis. The patient was a 61-year-old woman who had a radical mastectomy for breast cancer. Based on clinical evidence, acute hyponatremia had set in only a few days before onset of symptoms. The patient's disease progressed in two stages and became more severe during slow hyponatremia correction after 9 days from onset. Diffusion MRI provided early evidence of neurological lesions. In spite of a therapeutic attempt, the patient died unexpectedly 18 days after onset of her neurological disease due to massive pulmonary embolism. Histologically, our findings confirmed that the major features of central pontine myelinolysis in the acute stage are demyelination, the presence of large amounts of macrophages with no lymphocytic inflammatory reaction, and moderate astrocytosis. It is interesting to note that a monotypic immunological reaction persisted 19 days after radiological demonstration of parenchymal alterations.

1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074), a novel gamma-secretase modulator, reduces brain beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease without causing peripheral toxicity.

Some nonsteroidal anti-inflammatory drugs has been shown to allosterically modulate the activity of gamma-secretase, the enzymatic complex responsible for the formation of beta-amyloid (Abeta). 1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074) is a new gamma-secretase modulator, devoid of anticyclooxygenase (COX) and Notch-interfering activities in vitro. We evaluated the effects of chronic CHF5074 treatment on brain Abeta pathology in Tg2576 transgenic mice. Twenty-eight animals of 9.5 to 10.5 months of age received CHF5074-medicated diet (375 ppm) or standard diet for 17 weeks. Compared with controls, CHF5074 treatment significantly reduced the area occupied by plaques and the number of plaques in cortex (-52.2 +/- 5.6%, p = 0.0003 and -48.9 +/- 6.6%, p = 0.0004, respectively) and hippocampus (-76.7 +/- 6.4%, p = 0.004 and -66.2 +/- 10.3%, p = 0.037, respectively). Biochemical analysis confirmed the histopathological measures, with CHF5074-treated animals showing reduced total brain Abeta40 (-49.2 +/- 9.2%, p = 0.017) and Abeta42 (-43.5 +/- 9.7%, p = 0.027) levels. In a human neuroglioma cell line expressing Swedish mutated form of amyloid precursor protein (H4swe), CHF5074 reduced Abeta42 and Abeta40 secretion, with an IC50 of 3.6 and 18.4 microM, respectively, values consistent with those measured in the brain of the CHF5074-treated Tg2576 mice (6.4 +/- 0.4 microM). At 5 microM, no effects were observed on Notch intracellular cleavage in human embryonic kidney 293swe cells. CHF5074 was well tolerated by Tg2576 mice. No abnormal findings were observed upon histopathological examination of the gastrointestinal tract, indicating the absence of COX-related toxicity. Semiquantitative histochemical evaluation of goblet cells in the ileum of vehicle- and CHF5074-treated animals yielded similar results, suggesting no effects on Notch pathway. CHF5074 is therefore a promising therapeutic agent for Alzheimer's disease.

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide.

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.

Environmental exposure to trace elements and risk of amyotrophic lateral sclerosis: a population-based case-control study.

We analyzed the association between the environmental exposure to trace elements and the risk of sporadic amyotrophic lateral sclerosis (ALS) in a population-based case-control study in the Emilia-Romagna region in northern Italy. We evaluated exposure to selected trace elements by measuring toenail concentrations of the same by means of inductively coupled plasma optical spectrometry and instrumental neutron activation analysis. The final number enrolled in the study was 22 patients and 40 controls. Disease progression, assessed through a clinical score, was generally unassociated with toenail trace element levels, with the exception of an inverse relation with zinc and selenium content and a direct correlation with copper concentration. In logistic regression analysis, we found no evidence of an association between ALS risk and toenail content of cadmium, lead, copper, zinc, manganese, selenium, chromium, cobalt, iron, and aluminum. This investigation does not suggest a major role in sporadic ALS etiology of environmental exposure to these trace elements, though results for zinc, selenium, and copper should be evaluated with caution due to the potential limitations of toenails as biomarkers of chronic exposure in patients.