Down-regulation of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumour-infiltrating cells, and liver damage but accelerates tumour formation in nude mice

Fas-L (CD95L, APO-1L) expresses in a variety of tumours and has been proposed to play a role in tumour formation and metastasis. The contribution of Fas-L to tumour growth, however, is not conclusive especially in systems using cells with over-expressed Fas-L. In this study we down-regulated the expression o Fas-L in human glioma cells by a hammerhead ribozyme (Fas-Lribozyme) targeting against Fas-L mRNA. Fas-Lribozyme-carrying cells exhibited slightly enhanced growth rate and less degree of spontaneous apoptosis in vitro as compared with vector controls. In nude mice, Fas-Lribozyme-carrying cells grew faster with lesser apoptosis, formed bigger tumour with significantly fewer infiltrating cells in the tumour area, and triggered relatively milder tumour-associated liver damage than vector controls did. Thus, down-regulation of Fas-L not only improved viability of glioma cells but also reduces local immune responses that may consequently affect tumour formation. Taken together, our findings imply that endogenous expression of Fas-L in malignant cells is not always growth promoting. © 2001 Cancer Research Campaign  http://www.bjcancer.co

Raising children on a vegan diet: Parents’ opinion on problems in everyday life

A growing number of Italian families are adopting a vegan diet (VD) for their offspring from infancy for various reasons, with health benefits and ethics being the most common reasons. Barriers to effective communication with primary care pediatricians (PCPs) are perceived by many parents and, depending on the actors involved and the environment, a VD may affect social interactions in everyday life. A national cross‐sectional survey was conducted between July and September 2020. Parents of children following a VD completed an online questionnaire. Data from 176 Italian parents were collected. About 72% (71.8%) of the children included in this study had been on a VD since weaning. Parents did not inform their primary care pediatricians (PCP) about the VD in 36.2% of the cases. In 70.8% of the cases, PCPs were perceived as skeptical or against a VD. About 70% (71.2%) of the parents relied on medical dietitians, and 28.2% on nutritionists/dietitians for dietary counseling. Parents administered an individual B12 supplement in 87.2% of the cases. To the best of our knowledge, this survey is the first which explores the relationship between vegan parents and their PCPs, the parental management of their children’s diet and problems regarding the implementation of a VD in everyday life

A two-stage genome-wide association study of sporadic amyotrophic lateral sclerosis

The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown, but genetic factors are thought to play a significant role in determining susceptibility to motor neuron degeneration. To identify genetic variants altering risk of ALS, we undertook a two-stage genome-wide association study (GWAS): we followed our initial GWAS of 545 066 SNPs in 553 individuals with ALS and 2338 controls by testing the 7600 most associated SNPs from the first stage in three independent cohorts consisting of 2160 cases and 3008 controls. None of the SNPs selected for replication exceeded the Bonferroni threshold for significance. The two most significantly associated SNPs, rs2708909 and rs2708851 [odds ratio (OR) = 1.17 and 1.18, and P-values = 6.98 x 10–7 and 1.16 x 10–6], were located on chromosome 7p13.3 within a 175 kb linkage disequilibrium block containing the SUNC1, HUS1 and C7orf57 genes. These associations did not achieve genome-wide significance in the original cohort and failed to replicate in an additional independent cohort of 989 US cases and 327 controls (OR = 1.18 and 1.19, P-values = 0.08 and 0.06, respectively). Thus, we chose to cautiously interpret our data as hypothesis-generating requiring additional confirmation, especially as all previously reported loci for ALS have failed to replicate successfully. Indeed, the three loci (FGGY, ITPR2 and DPP6) identified in previous GWAS of sporadic ALS were not significantly associated with disease in our study. Our findings suggest that ALS is more genetically and clinically heterogeneous than previously recognized. Genotype data from our study have been made available online to facilitate such future endeavors

Broadening the phenotype of TARDBP mutations: the TARDBP Ala382Thr mutation and Parkinson’s disease in Sardinia

Mutations in the TARDBP gene are a cause of autosomal dominant amyotrophic lateral sclerosis (ALS) and of frontotemporal lobar degeneration (FTLD), but they have not been found so far in patients with Parkinson’s disease (PD). A founder TARDBP mutation (p.Ala382Thr) was recently identified as the cause of ~30% of ALS cases in Sardinia, a Mediterranean genetic isolate. We studied 327 consecutive Sardinian patients with clinically diagnosed PD (88 familial, 239 sporadic) and 578 Sardinian controls. One family with FTLD and parkinsonism was also included. The p.Ala382Thr heterozygous mutation was detected in eight unrelated PD patients (2.5%). The three patients from the FTLD/parkinsonism family also carried this mutation. Within the control group, there were three heterozygous mutation carriers. During follow-up, one of these individuals developed motoneuron disease and another, a rapidly progressive dementia; the third remains healthy at the age of 79 but two close relatives developed motoneuron disease and dementia. The eight PD patients carrying the p.Ala382Thr mutation had all sporadic disease presentation. Their average onset age was 70.0 years (SD 9.4, range 51–79), which is later but not significantly different from that of the patients who did not carry this mutation. In conclusion, we expand the clinical spectrum associated with TARDBP mutations to FTLD with parkinsonism without motoneuron disease and to clinically definite PD. The TDP-43 protein might be directly involved in a broader neurodegenerative spectrum, including not only motoneuron disease and FTLD but also PD

Proteostasis and ALS: Protocol for a phase II, randomised, double-blind, placebo-controlled, multicentre clinical trial for colchicine in ALS (Co-ALS)

Introduction: Disruptions of proteasome and autophagy systems are central events in amyotrophic lateral sclerosis (ALS) and support the urgent need to find therapeutic compounds targeting these processes. The heat shock protein B8 (HSPB8) recognises and promotes the autophagy-mediated removal of misfolded mutant SOD1 and TDP-43 fragments from ALS motor neurons (MNs), as well as aggregating species of dipeptides produced in C9ORF72-related diseases. In ALS-SOD1 mice and in human ALS autopsy specimens, HSPB8 is highly expressed in spinal cord MNs that survive at the end stage of disease. Moreover, the HSPB8-BAG3-HSP70 complex maintains granulostasis, which avoids conversion of dynamic stress granules (SGs) into aggregation-prone assemblies. We will perform a randomised clinical trial (RCT) with colchicine, which enhances the expression of HSPB8 and of several autophagy players, blocking TDP-43 accumulation and exerting crucial activities for MNs function. Methods and analysis: Colchicine in amyotrophic lateral sclerosis (Co-ALS) is a double-blind, placebo-controlled, multicentre, phase II RCT. ALS patients will be enrolled in three groups (placebo, colchicine 0.01 mg/day and colchicine 0.005 mg/day) of 18 subjects treated with riluzole; treatment will last 30 weeks, and follow-up will last 24 weeks. The primary aim is to assess whether colchicine decreases disease progression as measured by ALS Functional Rating Scale - Revised (ALSFRS-R) at baseline and at treatment end. Secondary aims include assessment of (1) safety and tolerability of Colchicine in patiets with ALS; (2) changes in cellular activity (autophagy, protein aggregation, and SG and exosome secretion) and in biomarkers of disease progression (neurofilaments); (3) survival and respiratory function and (4) quality of life. Preclinical studies with a full assessment of autophagy and neuroinflammation biomarkers in fibroblasts, peripheral blood mononuclear cells and lymphoblasts will be conducted in parallel with clinic assessment to optimise time and resources. Ethics and dissemination: The study protocol was approved by the Ethics Committee of Area Vasta Emilia Nord and by Agenzia Italiana del Farmaco (EUDRACT N.2017-004459-21) based on the Declaration of Helsinki. This research protocol was written without patient involvement. Patients' association will be involved in disseminating the study design and results. Results: will be presented during scientific symposia or published in scientific journals

Cystatin C: A Candidate Biomarker for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease characterized by progressive motor neuron degeneration. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective treatments. Reliable panels of diagnostic, surrogate, and prognostic biomarkers are needed to accelerate disease diagnosis and expedite drug development. The cysteine protease inhibitor cystatin C has recently gained interest as a candidate diagnostic biomarker for ALS, but further studies are required to fully characterize its biomarker utility. We used quantitative enzyme-linked immunosorbent assay (ELISA) to assess initial and longitudinal cerebrospinal fluid (CSF) and plasma cystatin C levels in 104 ALS patients and controls. Cystatin C levels in ALS patients were significantly elevated in plasma and reduced in CSF compared to healthy controls, but did not differ significantly from neurologic disease controls. In addition, the direction of longitudinal change in CSF cystatin C levels correlated to the rate of ALS disease progression, and initial CSF cystatin C levels were predictive of patient survival, suggesting that cystatin C may function as a surrogate marker of disease progression and survival. These data verify prior results for reduced cystatin C levels in the CSF of ALS patients, identify increased cystatin C levels in the plasma of ALS patients, and reveal correlations between CSF cystatin C levels to both ALS disease progression and patient survival

Rapamycin treatment for amyotrophic lateral sclerosis protocol for a phase II randomized, double-blind, placebo-controlled, multicenter, clinical trial (RAP-ALS trial)

Introduction: Misfolded aggregated proteins and neuroinflammation significantly contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, hence representing therapeutic targets to modify disease expression. Rapamycin inhibits mechanistic target of Rapamycin (mTOR) pathway and enhances autophagy with demonstrated beneficial effects in neurodegeneration in cell line and animal models, improving phenotype in SQSTM1 zebrafish, in Drosophila model of ALS-TDP, and in the TDP43 mouse model, in which it reduced neuronal loss and TDP43 inclusions. Rapamycin also expands regulatory T lymphocytes (Treg) and increased Treg levels are associated with slow progression in ALS patients. Therefore, we planned a randomized clinical trial testing Rapamycin treatment in ALS patients. Methods: RAP-ALS is a phase II randomized, double-blind, placebo-controlled, multicenter (8 ALS centers in Italy), clinical trial. The primary aim is to assess whether Rapamycin administration increases Tregs number in treated patients compared with control arm. Secondary aims include the assessment of safety and tolerability of Rapamycin in patients with ALS; the minimum dosage to have Rapamycin in cerebrospinal fluid; changes in immunological (activation and homing of T, B, NK cell subpopulations) and inflammatory markers, and on mTOR downstream pathway (S6RP phosphorylation); clinical activity (ALS Functional Rating Scale-Revised, survival, forced vital capacity); and quality of life (ALSAQ40 scale). Discussion: Rapamycin potentially targets mechanisms at play in ALS (i.e., autophagy and neuroinflammation), with promising preclinical studies. It is an already approved drug, with known pharmacokinetics, already available and therefore with significant possibility of rapid translation to daily clinics. Findings will provide reliable data for further potential trials. Ethics and dissemination: The study protocol was approved by the Ethics Committee of Azienda Ospedaliero Universitaria of Modena and by the Ethics Committees of participating centers (Eudract n. 2016-002399-28) based on the Helsinki declaration