Impact of highly active immunotherapy on acute and chronic neuroinflammation in aggressive multiple sclerosis

Background: Despite the use of new high efficacy therapies, complete disease remission is elusive in most people with multiple sclerosis (MS). This is particularly relevant for patients affected by aggressive MS, who deal with frequent relapses and accelerated accrual of irreversible disability. No evidence-based criteria exist to guide the choice on the best treatment approach in these patients. It is therefore urgent to collect data on available therapies for aggressive MS, analyzing their onset of action, the intensity of their immunosuppressive effects and their long-term clinical outcomes. Given that chronic, smoldering inflammation behind an intact blood-brain barrier has been identified as one of the most important drivers of disability progression, it is of fundamental importance to evaluate the impact of therapies on compartmentalized chronic neuroinflammation. This is particularly true for newer treatments, including hematopoietic stem cell transplantation (AHSCT), which is currently under investigation as a treatment strategy for aggressive MS. Aims: Against this background, the overarching aims of this thesis project were to: 1) Assess the clinical and MRI outcomes of patients with aggressive MS treated with different highly active immunotherapies 2) Investigate the impact of different highly active immunotherapies on chronic inflammation For the first aim of this project, we performed three different separate studies evaluating the impact of (i) AHSCT, (ii) alemtuzumab and (iii) ocrelizumab on clinical and MRI outcomes in patients presenting an aggressive form of MS. For the second aim, we performed a longitudinal prospective quantitative MRI study evaluating the impact of AHSCT and other highly active therapies on acute and chronic inflammation assessed with quantitative susceptibility mapping (QSM) in aggressive MS patients. Then, aiming at comprehensively detect the smoldering inflammatory component in MS lesions, we performed a combined quantitative MRI study investigating the relationship between axonal integrity, myelin content and iron deposition in the entire spectrum of MS lesions. Results: First, we showed that AHSCT prevented disability progression and inflammatory disease activity in most patients affected by relapsing-remitting MS and that these effects last for more than a decade. We also demonstrated that AHSCT significantly reduced the risk of relapses and MRI activity, allowing complete disease remission in a higher proportion of patients compared with alemtuzumab. Similar results were observed in patients treated with ocrelizumab, although we observed some evidence of persistent MRI activity in the first year of treatment. We showed that chronic smoldering inflammation, detected by QSM as paramagnetic rim lesions, is frequent in aggressive MS, being detectable in 80% of patients. Our preliminary results show that chronic active, paramagnetic rim lesions tend to persist over time, despite the use of highly active immunosuppression, including AHSCT. However, we found that some lesions exhibit changes in the intensity and the distribution of QSM signal over time, which could be related, at least in part, to highly active CNS penetrant drugs treatment used in these patients. Lastly, with a multimodal MRI approach we distinguished different types of MS lesions (hypo-isointense, homogeneous hyperintense, inhomogeneous hyperintense and paramagnetic rim), which were characterized by increasing degrees of axonal and myelin disruption. Discussion: Aggressive MS is characterized by a great amount of acute and chronic neuroinflammation, which can be detected and monitored in vivo by quantitative MRI. Among high efficacy immunotherapies, AHSCT has the potential to control neuroinflammation allowing long-lasting disease remission

Mechanical and Transport Phenomena in Advanced Pollutants Containment Systems. A theoretical and experimental study

Geotechnical engineers and hydrologists have dedicated most of their efforts devoted to problems concerning environmental protection and human health since the 1970’s, by studying the interaction between soils and polluting substances. Such remarkable impetus of studies emerged mainly from the need to manage the disposal of radioactive materials produced by nuclear stations with solutions which would be able to guarantee an adequate level of safety in the long term (Daniel, 1993). Scientific community was soon called on to study industrial waste landfills and pollution of the sub-soil deriving from materials produced by chemical and petro-chemical industries: in particular, the main problem to solve was that of designing adequate pollutant barrier systems in order to limit contaminant discharge to groundwater and related migration in the subsoil (Benson, 2000). Initially, such barriers were made up by mineral type materials (drainage layers in sand and gravel, compacted clay liners, soil-bentonite and cement-bentonite cutoff-walls), since their properties for long-term use were considered sufficiently durable. Clay liners and clay caps were used respectively in order to provide isolation of waste leachate from the subsoil (in the case of liners), or to guarantee long-term control of percolation into the waste and control leachate generation (in the case of covers). The design of pollutant barrier systems has been innovatively modified since the mid-1980’s with the introduction of the so-called Geosynthetic Clay Liners (GCLs), which are defined as factory-manufactured hydraulic or gas barriers consisting of a layer of bentonite or other very low permeability material supported by geotextiles and/or geomembranes, mechanically held together by needling, stitching or chemical adhesives (Koerner and Koerner, 2010). The hydraulic resistance of conventional GCLs (i.e. which are not comprised of a geomembrane component) is attributed to the bentonite component of the GCL, which swells in the presence of water to form a tight sealing layer (Shackelford, 2007). GCLs have been utilized greatly as the lower portion of geomembrane/GCL composites in both landfill liners and final covers (e.g. Bouazza, 2002; Koerner, 2005); they have also been used in other containment applications and by themselves as single barrier systems when modified with a geofilm or polymer coating within the cover geotextiles. In most applications they have served as replacement materials for the more traditional compacted clay liners (CCLs) in cover systems or in bottom lining of waste containment facilities, since they present very low hydraulic conductivity to water and relatively low cost (Bouazza, 2002). GCLs have undergone great change since the 1990’s. Engineers had to explore new theoretical problems, such as the study of solute diffusion phenomena in soils and chemical osmosis in bentonites, since hydraulic performances of GCLs are greatly affected by the chemical composition of the environment surrounding the barrier (e.g. they can be worsen by a simple variation of the chemical and physical boundary conditions), and depend on the swelling and osmotic behaviour which bentonite, characterized by a high content in montmorillonite, may exhibit. Moreover, new specific design methodologies have been developed, whose distinctive features derive from the specific nature of the objective to reach, of the materials employed, of the boundary conditions and of the reference regulations; an important example of such new methodologies is represented by risk analysis. The research project developed during the PhD has been focused on bentonite barriers which are designed both in urban waste landfill, hazardous or radioactive wastes final disposal. The theoretical and experimental study has had the aim of studying the mechanical and chemico-osmotic behaviour of bentonite in contact with standard (i.e. de-ionized water, DW) and non standard liquids (i.e. sodium chloride solutions), acting on its state parameters, chemical composition, and boundary conditions at installation. The whole laboratory activity, comprising oedometer and chemico-osmotic tests, was carried out at “Politecnico di Torino”, in the Disaster Planning Laboratory (DIPLAB) of the Department of Structural, Geotechnical and Building Engineering (DISEG). The contents of the 3-year PhD research are reported in this thesis and are resumed in the short summary reported below: Chapter 1 – Bentonite barriers This chapter is aimed at introducing the main topics concerning the mineralogical, chemical and physical description of sodium bentonite; swelling and osmotic phenomena are also presented. Particular attention is devoted to the engineering properties which can be evaluated in order to characterize bentonite, in particular of Geosynthetic Clay Liners, for geoenvironmental applications: in this regard the most important characteristics of sodium bentonite which was employed for the PhD research activity are resumed. A propaedeutic study on mechanical behaviour of bentonites, referring in particular to the swelling phenomenon, is also described. Finally the main aspects and issues concerning practical application and proper use of Geosynthetic Clay Liners and Clay membrane barriers in general are introduced. Chapter 2 – Theoretical models for mechanical and osmotic behaviour of sodium bentonites The electric interaction between montmorillonite particles, which represent the main mineralogical component of bentonite, and the ions in pore solution determines macroscopic phenomena which cannot be modelled on the basis of the classical theories used to describe the movement of water and solutes through porous media. This chapter is focused on the theoretical approaches which can be adopted in order to model transport properties of bentonites and the related aspects of their mechanical behaviour. The phenomenological and physical theoretical approaches are complementary and represent a very useful tool for experimental data interpretation, in order to reduce the number of tests to be performed and simulate GCLs behaviour in applications, even in the long-term, under boundary conditions different from those adopted in laboratory. Chapter 3 – Mechanical and swelling behaviour of bentonites In the Paper included in this chapter, titled “MECHANICAL AND SWELLING BEHAVIOUR OF SODIUM BENTONITES IN EQUILIBRIUM WITH LOW MOLARITY NaCl SOLUTIONS UNDER OEDOMETRIC CONDITIONS”, a theoretical and experimental study on mechanical and swelling behaviour of tested sodium bentonite is presented. Several oedometer tests (i.e. with a conventional apparatus and a new testing device) were performed on sodium bentonite specimens in equilibrium with NaCl 0.01 M concentrated solutions in order to measure a phenomenological parameter, i.e. the swelling pressure, at different void ratios. The obtained experimental results have been interpreted on the basis of the theoretical model, by assuming that the microscopic deviations of the pore solution state variables from their average values are negligible: in such a way, it was possible to interpret the macroscopic behaviour on the basis of the physical and chemical properties of the bentonite mineralogical components, and, thus, characterize the microstructure of the material. In the last part of the chapter other oedometer tests are presented, with the aim of analysing the effect of different salt concentrations of the equilibrium solution on the mechanical and swelling behaviour of sodium bentonite. Chapter 4 – Chemico-osmotic behaviour of bentonites Membrane behaviour represents a potential benefit in engineered clay-based barriers for geoenvironmental applications, especially if such barriers consist of sodium bentonite. A theoretical and experimental study on membrane behaviour of tested sodium bentonite is presented in the Paper included in this chapter, titled “INFLUENCE OF SPECIMEN POROSITY AND SODIUM CHLORIDE PORE SOLUTION CONCENTRATION ON CHEMICO-OSMOTIC BEHAVIOUR OF SODIUM BENTONITES”. The effects of porosity on two natural sodium bentonite specimens submitted to multiple-stage chemico-osmotic tests were investigated, by evaluating two phenomenological parameters that affect transport properties of bentonite, i.e. the chemico-osmotic reflection coefficient and the osmotic effective diffusion coefficient. The experimental results were compared to literature data and interpreted on the basis of the proposed theoretical approach, under the hypothesis that the microscopic deviations of the state variables from their average values are negligible. Even in this case, it was possible to interpret the macroscopic behaviour on the basis of the physical and chemical properties of the bentonite mineralogical components. In the last paragraph of the chapter, two further chemico-osmotic tests, by means of a new testing apparatus, are presented: experimental data are interpreted through the proposed theoretical framework and compared with previous results

Sound velocities and single-crystal elasticity of hydrous Fo90 olivine to 12 GPa

Nominally anhydrous minerals (NAMs) may contain significant amounts of water and constitute an important reservoir for mantle hydrogen. The colloquial term ‘water’ in NAMs is related to the presence of hydroxyl-bearing (OH-) point defects in their crystal structure, where hydrogen is bonded to lattice oxygen and is charge-balanced by cation vacancies. This hydrous component may therefore have substantial effects on the thermoelastic parameters of NAMs, comparable to other major crystal-chemical substitutions (e.g., Fe, Al). Assessment of water concentrations in natural minerals from mantle xenoliths indicates that olivine commonly stores ~0 – 200 ppm of water. However, the lack of samples originating from depths exceeding ~250 km coupled with the rapid diffusion of hydrogen in olivine at magmatic temperatures makes the determination of the olivine water content in the upper mantle challenging. On the other hand, numerous experimental data show that, at pressures and temperatures corresponding to deep upper mantle conditions, the water storage capacity of olivine increases to 0.2 – 0.5 wt.%. Therefore, determining the elastic properties of olivine samples with more realistic water contents for deep upper mantle conditions may help in interpreting both seismic velocity anomalies in potentially hydrous regions of Earth's mantle as well as the observed seismic velocity and density contrasts across the 410-km discontinuity. Here, we report simultaneous single-crystal X-ray diffraction and Brillouin scattering experiments at room temperature up to 11.96(2) GPa on hydrous (0.20(3) wt.% H2O) Fo90 olivine to assess its full elastic tensor, and complement these results with a careful re-analysis of all the available single-crystal elasticity data from the literature for anhydrous Fo90 olivine. While the bulk (K) and shear (G) moduli of hydrous Fo90 olivine are virtually identical to those of the corresponding anhydrous phase, their pressure derivatives K´ and G´ are slightly larger, although consistent within mutual uncertainties. We then defined linear relations between the water concentration in Fo90 olivine, the elastic moduli and their pressure derivatives, which were then used to compute the sound velocities of Fo90 olivine with higher degrees of hydration. Even for water concentrations as high as 0.5 wt.%, the sound wave velocities of hydrous and anhydrous olivines were found to be identical within uncertainties at pressures corresponding to the base of the upper mantle. Contrary to previous claims, our data suggest that water in olivine is not seismically detectable, at least for contents consistent with deep upper mantle conditions. In addition to that, our data reveal that the hydration of olivine is unlikely to be a key factor in reconciling seismic velocity and density contrasts across the 410-km discontinuity with a pyrolitic mantle

Relationship between retinal inner nuclear layer, age, and disease activity in progressive MS

ObjectiveTo investigate whether inner nuclear layer (INL) thickness as assessed with optical coherence tomography differs between patients with progressive MS (P-MS) according to age and disease activity.MethodsIn this retrospective longitudinal analysis, differences in terms of peripapillary retinal nerve fiber layer (pRNFL), ganglion cell layer + inner plexiform layer (GCIPL), INL and T1/T2 lesion volumes (T1LV/T2LV) were assessed between 84 patients with P-MS and 36 sex- and age-matched healthy controls (HCs) and between patients stratified according to age (cut-off: 51 years) and evidence of clinical/MRI activity in the previous 12 monthsResultspRNFL and GCIPL thickness were significantly lower in patients with P-MS than in HCs (p = 0.003 and p < 0.0001, respectively). INL was significantly thicker in patients aged < 51 years compared to the older ones and HCs (38.2 vs 36.5 and 36.7 m; p = 0.038 and p = 0.04, respectively) and in those who presented MRI activity (new T2/gadolinium-enhancing lesions) in the previous 12 months compared to the ones who did not and HCs (39.5 vs 36.4 and 36.7 m; p = 0.003 and p = 0.008, respectively). Recent MRI activity was significantly predicted by greater INL thickness (Nagelkerke R2 0.36, p = 0.001).ConclusionsINL thickness was higher in younger patients with P-MS with recent MRI activity, a criterion used in previous studies to identify a specific subset of patients with P-MS who best responded to disease-modifying treatment. If this finding is confirmed, we suggest that INL thickness might be a useful tool in stratification of patients with P-MS for current and experimental treatment choic

A Two Alternative Forced Choice Method for Assessing Vibrotactile Discrimination Thresholds in The Lower Limb

The development of an easy to implement, quantitative measure to examine vibration perception would be useful for future application in clinical settings. Vibration sense in the lower limb of younger and older adults was examined using the method of constant stimuli (MCS) and the two-alternative forced choice paradigm. The focus of this experiment was to determine an appropriate stimulation site on the lower limb (tendon versus bone) to assess vibration threshold and to determine if the left and right legs have varying thresholds. Discrimination thresholds obtained at two stimulation sites in the left and right lower limbs showed differences in vibration threshold across the two ages groups, but not across sides of the body nor between stimulation sites within each limb. Overall, the MCS can be implemented simply, reliably, and with minimal time. It can also easily be implemented with low-cost technology. Therefore, it could be a good candidate method to assess the presence of specific deep sensitivity deficits in clinical practice, particularly in populations likely to show the onset of sensory deficits

Different MRI patterns in MS worsening after stopping fingolimod

Objective To analyze MRI images in patients with MS who experienced worsening of neurologic status (WNS) after stopping fingolimod (FTY).MethodsIn this retrospective study, demographic, clinical, and radiologic data of patients with MS who experienced WNS after stopping FTY were retrospectively collected. We introduced the "\u3b4Expanded Disability Status Scale (EDSS)-ratio" to identify patients who, after FTY withdrawal, showed an inflammatory flare-up exceeding the highest lifetime disease activity level. Patients with \u3b4EDSS-ratio > 1 were enrolled in the study.ResultsEight patients were identified. The mean (SD) age of the 8 (7 female) patients was 35.3 (4.9) years. The mean FTY treatment duration was 3.1 (0.8) years. The mean FTY discontinuation-WNS interval was 4 (0.9) months. The 4 patients with \u3b4EDSS-ratio 65 2 developed severe monophasic WNS (EDSS score above 8.5), characterized by clinical features and MRI findings not typical of MS, which we classified as "tumefactive demyelination pattern" (TDL) and "Punctuated pattern" (PL). Conversely, patients whose \u3b4EDSS-ratio was between 1 and 2 had clinical features and brain MRI compatible with a more typical, even if aggressive, MS relapse. In patients with TDL and PL, the flare-up of inflammatory activity led to severe tissue damage resulting in T2 but also T1 lesion volume increase at 6-month follow-up.ConclusionsPeculiar MRI features (TDL and PL), different from a typical MS flare-up, might occur in some patients who experienced WNS after stopping FTY. Further studies, also involving immunologic biomarkers, are necessary to investigate TDL or PL pathophysiology