Time to generalization and prediction of survival in patients with amyotrophic lateral sclerosis: a retrospective observational study.

BACKGROUND AND PURPOSE: A strong association between time to generalization (TTG), considered as the time of spreading of the clinical signs from spinal or bulbar localization to both, and survival was recently identified in patients with amyotrophic lateral sclerosis (ALS). Thus, TTG may be used as an early to intermediate end-point in survival studies. The aim of the present study was to test TTG as a predictor of survival in ALS. METHODS: This was an observational retrospective study of ALS patients from a tertiary referral centre over a 5-year follow-up period. RESULTS: In 212 ALS patients, TTG was associated with time to death/tracheostomy [R 0.62, 95% confidence interval (CI) 0.53-0.70; P < 0.001]. In a time-to-event analysis, longer TTG resulted in lower risk to reach a composite outcome (death or tracheostomy) both in univariate [hazard ratio (HR) 0.98, 95% CI 0.97-0.99] and multivariate Cox analyses (HR 0.98, 95% CI 0.96-0.99). TTG predicted death/tracheostomy at 4 years (C-statistic 0.58; 95% CI 0.53-0.63) and at 5 years (C-statistic 0.58; 95% CI 0.53-0.62). CONCLUSIONS: Based on the present results from a large clinical cohort, TTG may be used as a new early to intermediate end-point to describe the ALS natural history. TTG may be potentially useful as a new primary outcome measure for clinical trials.

Epigenomic profiling in visceral white adipose tissue of offspring of mice exposed to late gestational sleep fragmentation.

BACKGROUND: Sleep fragmentation during late gestation (LG-SF) is one of the major perturbations associated with sleep apnea and other sleep disorders during pregnancy. We have previously shown that LG-SF induces metabolic dysfunction in offspring mice during adulthood. OBJECTIVES: To investigate the effects of late LG-SF on metabolic homeostasis in offspring and to determine the effects of LG-SF on the epigenome of visceral white adipose tissue (VWAT) in the offspring. METHODS: Time-pregnant mice were exposed to LG-SF or sleep control during LG (LG-SC) conditions during the last 6 days of gestation. At 24 weeks of age, lipid profiles and metabolic parameters were assessed in the offspring. We performed large-scale DNA methylation analyses using methylated DNA immunoprecipitation (MeDIP) coupled with microarrays (MeDIP-chip) in VWAT of 24-week-old LG-SF and LG-SC offspring (n=8 mice per group). Univariate multiple-testing adjusted statistical analyses were applied to identify differentially methylated regions (DMRs) between the groups. DMRs were mapped to their corresponding genes, and tested for potential overlaps with biological pathways and gene networks. RESULTS: We detected significant increases in body weight (31.7 vs 28.8 g; P=0.001), visceral (642.1 vs 497.0 mg; P=0.002) and subcutaneous (293.1 vs 250.1 mg; P=0.001) fat mass, plasma cholesterol (110.6 vs 87.6 mg dl(-1); P=0.001), triglycerides (87.3 vs 84.1 mg dl(-1); P=0.003) and homeostatic model assessment-insulin resistance values (8.1 vs 6.1; P=0.007) in the LG-SF group. MeDIP analyses revealed that 2148 DMRs (LG-SF vs LG-SC; P<0.0001, model-based analysis of tilling-arrays algorithm). A large proportion of the DMR-associated genes have reported functions that are altered in obesity and metabolic syndrome, such as Cartpt, Akt2, Apoe, Insr1 and so on. Overrepresented pathways and gene networks were related to metabolic regulation and inflammatory response. CONCLUSIONS: Our findings show a major role for epigenomic regulation of pathways associated with the metabolic processes and inflammatory responses in VWAT. LG-SF-induced epigenetic alterations may underlie increases in the susceptibility to obesity and metabolic syndrome in the offspring.

Cerebrospinal fluid neurofilament light chain levels: marker of progression to generalized amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: To evaluate whether cerebrospinal fluid (CSF) neurofilament light chain (NFL) levels could predict the time to generalization (TTG) in amyotrophic lateral sclerosis (ALS). METHODS: Cerebrospinal fluid NFL levels of 37 cases of sporadic ALS were measured and the time of symptom spreading from spinal or bulbar localization to both (TTG) was evaluated in all patients. RESULTS: Kaplan-Meier analysis showed a short TTG in patients with high NFL levels (log-rank test chi-squared = 19.4, P < 0.0001). In a multivariate regression model patients with NFL levels above the median had an eight-fold higher risk of generalization (adjusted hazard ratio 7.9, 95% confidence interval 2.9-21.4, P < 0.0001) compared with those with NFL levels below the median. CONCLUSIONS: This study shows that in sporadic ALS NFL, a marker of neurodegeneration, is correlated with TTG, a clinical intermediate parameter of survivorship.

Brain and spinal cord atrophy in NMOSD and MOGAD: Current evidence and future perspectives.

Neuromyelitis optica spectrum disorder (NMOSD) is a severe form of inflammation of the central nervous system (CNS) including acute myelitis, optic neuritis and brain syndrome. Currently, the classification of NMOSD relies on serologic testing, distinguishing between seropositive or seronegative anti-aquaporin-4 antibody (AQP4) status. However, the situation has recently grown more intricate with the identification of patients exhibiting the NMOSD phenotype and myelin oligodendrocyte glycoprotein antibodies (MOGAD). NMOSD is primarily recognized as a relapsing disorder; MOGAD can manifest with either a monophasic or relapsing course. Significant symptomatic inflammatory CNS injuries with stability in clinical findings outside the acute phase are reported in both diseases. Nevertheless, recent studies have proposed the existence of a subclinical pathological process, revealing longitudinal changes in brain and spinal cord atrophy. Within this context, we summarise key studies investigating brain and spinal cord measurements in adult NMOSD and MOGAD. We also explore their relationship with clinical aspects, highlight differences from multiple sclerosis (MS), and address future challenges. This exploration is crucial for determining the presence of chronic damage processes, enabling the customization of therapeutic interventions irrespective of the acute phase of the disease.

Elevated cerebrospinal fluid neurofilament light levels in patients with amyotrophic lateral sclerosis: a possible marker of disease severity and progression.

BACKGROUND: To date there are no biomarkers with proven reliability as a measure of disease burden in amyotrophic lateral sclerosis (ALS). The aim of our study is to assess the neurofilament light chain (NFL) in cerebrospinal fluid (CSF) samples as a measure of disease activity and progression in ALS. METHODS: Thirty-seven consecutive patients with ALS, 25 with chronic inflammatory demyelinating polyneuropathy and 21 with other neurodegenerative diseases were evaluated. CSF NFL levels were assayed by two-site solid-phase sandwich ELISA. In patients with ALS, neurological status was assessed by the revised ALS Functional Rating Scale (ALSFRS-r) and the Medical Research Council scale, and the progression of the disease was evaluated using the 'diagnostic delay' and the 'progression rate'. RESULTS: Cerebrospinal fluid NFL levels were higher in ALS cases than in controls (P < 0.0001). Using receiver operating curve analysis, an optimal NFL cut-off of 1981 ng/l discriminated between patients with ALS and neurological controls, with a sensitivity of 78.4% and specificity of 72.5%. Multivariate logistic regression confirmed the association between CSF NFL levels and the presence of ALS (age and sex adjusted odds ratio for ALS 8.9; 95% CI 3.1-25.8; P < 0.0001). In ALS, CSF NFL negatively correlated with the diagnostic delay (P < 0.0001) and the ALSFRS-r (P = 0.014) and positively with the progression rate (P < 0.0001). CONCLUSIONS: High CSF NFL levels were found in patients with ALS, reflecting the burden of neurodegeneration. The significant relation between CSF NFL levels and disease progression suggests that NFL may be a useful marker of disease activity and progression in ALS.

Pharmacological aspects and neurological speech therapy: target of dysphony, dysarthria and dysphagia.

The word dysphagy was suggested by Nicolatopoulos (1907) and derives from the ancient Greek "duz", which means "difficulty" and "katapinein", which means "to swallow". Generally, the dysphagia is defined on the basis of its origin: oral, pharynx and oesophagus, otherwise by its mechanical or neurological aetiology. The symptoms are dependent on the nature of the lesions in the affected organs. The swallow is a complex motor sequence dependent on the coordinate contraction of the muscle of mouth, of larynx and of the oesophagus. The mechanical action of the swallow helps the liquid or solid food progression from mouth to stomach thanks to cooperation of 31 muscles and 5 cranial nerves and allows swallowing about 580 times approximately. The dysphagy in neurological diseases is mainly due to the following reasons. Increase of vascular cerebral disease, increase of population age and increase of road and work traumas. The difficulties in swallowing causes heavy social problems like meager diet, social isolation and worsening of quality of life. The speech rehabilitation requires the involvement of care givers through a re-educational program that takes place in two periods: the first of relaxation, and the second of restoration of phonodeglutition praxis.

Transcriptional regulation of liver-specific gene expression.

Liver-specific gene expression is regulated by four families of transcriptional activatory proteins that are not liver-specific, but are still restricted to a subset of tissues. The current opinion is that liver-specificity is a consequence of the combinatorial action of these factors, which could represent 'functional compartments' coexisting in the hepatocyte.

Transcription of the promoter of the rat NF-1 gene depends on the integrity of an Sp1 recognition site.

The transcription start site and promoter of the rat gene coding for the transcription factor NF-1 have been identified. The NF-1 promoter was fused to the chloramphenicol acetyltransferase-coding sequence, and the resulting plasmid was transcriptionally active in the HepG2 cell line. Footprinting and gel retardation analysis indicated that the transcription factor Sp1 binds to the NF-1 promoter. Mutants in the Sp1-binding site displayed a strong reduction in transcriptional activity.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

DNA-based selection and screening of peptide ligands.

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.

Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist.

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.

Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium.

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays.

Mutagenicity of diallate, sulfallate, and triallate and relationship between structure and mutagenic effects of carbamates used widely in agriculture.

In an investigation of the mutagenic properties of 20 carbamate herbicides and fungicides by use of the Salmonella/microsome mutagenicity test as developed by Ames et al. (Mutation Res., 31: 347-364, 1975), we have found that three thiocarbamate compounds, diallate, sulfallate and triallate, are mutagenic in the presence of a liver microsomal fraction on strains TA1535 and TA100. This indicates that the metabolic products of these thiocarbamates are causing base-pair substitutions. Since the 2-chloro-allyl group is common to the three mutagenic compounds but is not common to the 17 nonmutagenic compounds, a metabolic derivative of this group is probably responsible for the mutagenic activity.

Mutagenicity of pesticides containing 1,3-dichloropropene.

In a systematic study of the mutagenic effect of chemical compounds used as pesticides, we found that D. D. soil fumigant and Telone are mutagenic. The test was performed using the bacterial tester strains following the procedure developed by Ames. The active principle of D. D. soil fumigant and Telone is a mixture of the cis and trans isomers of 1,3-dichloropropene. Both isomers are mutagenic in Salmonella strains TA 1535 and TA 100. 2,3-Dichloro-1-propene, a minor component (5%) of the commercial preparation Telone, was also found to be mutagenic in strains TA 1535 and TA 100. Mutagenesis of these tester strains is an indication of a base-pair substitution event causing a missense mutation. 1,3-Dichloropropene is widely used in agriculture all over the world. In Italy 2,187,100 kg were produced in 1972. In California over 1,000,000 kg of 1,3-dichloropropene-containing pesticides were used in 1971.

Coupling protein design and in vitro selection strategies: improving specificity and affinity of a designed beta-protein IL-6 antagonist.

The minibody is a designed small beta-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two beta-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for "stringent" selection such as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.

The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.

Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda.

We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda. cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immuno-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and VIII capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery.

A conformationally homogeneous combinatorial peptide library.

In search for a rational way to convert the information encoded in peptide structures into peptidomimetics, major progress could be made by coupling the power of selection methods, now enormously increased in number as a result of the development of combinatorial peptide libraries, with the rational design of structure-inducing templates for the selectable sequences. The availability of libraries of peptides with predetermined structure would enable selection-driven peptidomimetic design, whereby a conformational model for the peptide pharmacophore would be directly derived from the screening, allowing the design of a suitable non-peptidic scaffold to replace the peptide backbone. We describe here the first example of a conformationally homogeneous combinatorial peptide library, which yields ligands with the expected structure upon selection. The library was built by randomising five positions in the alpha-helical portion of a 26 amino acid Cys2His2 consensus "zinc-finger" motif. Since in zinc-fingers metal coordination and folding are coupled, in our library metal-dependent binding represents a built-in control against the selection of structurally undefined sequences. The alpha-helical library was produced as both fusion with the pVIII protein of filamentous phage and soluble peptides by chemical synthesis, the latter enabling the expansion of the selectable repertoire by the inclusion of non-coded amino acids. The two libraries were independently screened with the same receptor (a monoclonal IgA reactive against the lipopolysaccharide of the human pathogen Shigella flexneri), yielding a very similar consensus. In particular, the peptides defined by both methods showed very strong, zinc-dependent binding to the IgA. The geometrical arrangement of the side-chains of the selected peptide pharmacophore was shown by circular dichroism, Co(II)-complex absorption and high-resolution NMR to be structurally invariant with respect to the parent zinc-finger.

Identification of a novel type 1 diabetes-specific epitope by screening phage libraries with sera from pre-diabetic patients.

We used random peptide libraries displayed on phage to search for ligands to insulin dependent diabetes mellitus-related antibodies and were able to identify several candidate disease-related peptides. One of them, clone 92, showed a significant difference in the frequency of reactivity with the sera of patients and normal controls. Human immunoglobulins immunopurified on phage 92 specifically stained the islets on human pancreatic sections. When injected into rabbits, the selected peptide elicited antibodies that also stained human and rat pancreatic sections, with a pattern similar to that observed with immunoglobulins purified from the sera of patients. No reactivity was observed in other tissues. Our results indicate that the peptide identified in this work mimics a novel, diabetes-related self-antigen.