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Niloufar Zarghami,1 Alexandre A Khrapitchev,1 Francisco Perez-Balderas,1 Manuel Sarmiento Soto,1 James R Larkin,1 Luca Bau,2 Nicola R Sibson1 1Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK; 2Institute of Biomedical Engineering, Department of Engineering Sciences, University of Oxford, Oxford, UK Background: Molecular MRI is an evolving field of research with strong translational potential. Selection of the appropriate MRI sequence, field strength and contrast agent depend largely on the application. The primary aims of the current study were to: 1) assess the sensitivity of different MRI sequences for detection of iron oxide particles in mouse brain; 2) determine the effect of magnetic field strength on detection of iron oxide particles in vivo; and 3) compare the sensitivity of targeted microparticles of iron oxide (MPIO) or ultra-small superparamagnetic iron oxide (USPIO) for detection of vascular cell adhesion molecule-1 (VCAM-1) in vivo. Methods: Mice were injected intrastriatally with interleukin 1β to induce VCAM-1 expression on the cerebral vasculature. Subsequently, animals were injected intravenously with either VCAM-MPIO or VCAM-USPIO and imaged 1 or 13 hours post-injection, respectively. MRI was performed at 4.7, 7.0, or 9.4 T, using three different T2*-weighted sequences: single gradient echo 3D (GE3D), multi-gradient echo 3D (MGE3D) and balanced steady-state free precession 3D (bSSFP3D). Results: MGE3D yielded the highest signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) for the detection of iron oxide particles. All sequences showed a significant increase in SNR and CNR from 4.7 to 7.0 T, but no further improvement at 9.4 T. However, whilst targeted MPIO enabled sensitive detection of VCAM-1 expression on the cerebral vasculature, the long half-life (16.5 h vs 1.2 min) and lower relaxivity per particle (1.29×10–14 vs 1.18×10–9 Hz L/particle) of USPIO vs. MPIO rendered them impractical for molecular MRI. Conclusion: These findings demonstrate clear advantages of MPIO compared to USPIO for molecularly-targeted MRI, and indicate that the MGE3D sequence is optimal for MPIO detection. Moreover, higher field strengths (7.0/9.4 T) showed enhanced sensitivity over lower field strengths (4.7 T). With the development of biodegradable MPIO, these agents hold promise for clinical translation. Keywords: molecular imaging, preclinical MRI, VCAM-MPIO, VCAM-USPIO, multi gradient echo, bSSFP

Rachel Myers,1 Megan Grundy,2 Cliff Rowe,1 Christian M Coviello,1 Luca Bau,2 Philippe Erbs,3 Johann Foloppe,3 Jean-Marc Balloul,3 Colin Story,1 Constantin C Coussios,2 Robert Carlisle2 1OxSonics Ltd, The Magdalen Centre, 2BUBBL, IBME, Department of Engineering Science, University of Oxford, Oxford, UK; 3Transgene SA, Illkirch-Graffenstaden, France Abstract: The treatment of cancer using nanomedicines is limited by the poor penetration of these potentially powerful agents into and throughout solid tumors. Externally controlled mechanical stimuli, such as the generation of cavitation-induced microstreaming using ultrasound (US), can provide a means of improving nanomedicine delivery. Notably, it has been demonstrated that by focusing, monitoring and controlling the US exposure, delivery can be achieved without damage to surrounding tissue or vasculature. However, there is a risk that such stimuli may disrupt the structure and thereby diminish the activity of the delivered drugs, especially complex antibody and viral-based nanomedicines. In this study, we characterize the impact of cavitation on four different agents, doxorubicin (Dox), cetuximab, adenovirus (Ad) and vaccinia virus (VV), representing a scale of sophistication from a simple small-molecule drug to complex biological agents. To achieve tight regulation of the level and duration of cavitation exposure, a “cavitation test rig” was designed and built. The activity of each agent was assessed with and without exposure to a defined cavitation regime which has previously been shown to provide effective and safe delivery of agents to tumors in preclinical studies. The fluorescence profile of Dox remained unchanged after exposure to cavitation, and the efficacy of this drug in killing a cancer cell line remained the same. Similarly, the ability of cetuximab to bind its epidermal growth factor receptor target was not diminished following exposure to cavitation. The encoding of the reporter gene luciferase within the Ad and VV constructs tested here allowed the infectivity of these viruses to be easily quantified. Exposure to cavitation did not impact on the activity of either virus. These data provide compelling evidence that the US parameters used to safely and successfully delivery nanomedicines to tumors in preclinical models do not detrimentally impact on the structure or activity of these nanomedicines. Keywords: nanomedicine, antibody, virus, ultrasound, cavitation

This paper presents a detailed study on the structural, magnetic, and critical behavior and magnetocaloric effect of LaSrMnCuO prepared by solid-state reaction method. X-ray diffraction patterns indicate that Cu-doped manganite crystallizes to a single phase with a rhombohedral structure. Critical components β, γ, and δ were determined by using modified Arrott plot, Kouvel-Fisher method, and critical isotherm analyses. These components are consistent with their corresponding values and were validated by Widom scaling law and scaling theory. The deduced critical exponents ( β = 0.456, γ = 0.9912, and δ = 3.206) are also close to their corresponding theoretical values predicted by mean-field model, indicating that long-range interaction dominates the critical behavior of the LSMCO system. Spontaneous magnetization was determined by two methods. One method is based on magnetic entropy change under different magnetic fields, and the other method is based on classical extrapolation from the Arrott plot. The two methods are in excellent agreement, confirming the validity of the deduction of spontaneous magnetization using the magnetic entropy change. [ABSTRACT FROM AUTHOR]

We have investigated the structure, magnetic, and electrical properties, and the resistance switching effect (RSE) of La2NiO $_{{ {4+\delta }}} compounds with different excess oxygen levels of \delta = 0.004 - 0.031 . Our results showed the structure changes from tetragonal I4/mmm to orthorhombic Fmmm when reducing \delta value from 0.011 to 0.006. The M(H) curves measured at several temperatures exhibit a coexistence of the weak ferromagnetic and antiferromagnetic interactions. Two shoulders on M(T) curves related to the spin and charge ordering have also been observed. These ordering temperatures depend strongly on \delta ) and low ( RL ) values. The resistance change ratio of the samples defined as 100% \times [RH - RL]/RL is found to be about 18%–57%, which depends on the value of $\delta $ in the samples. [ABSTRACT FROM AUTHOR]

Animal-derived lung surfactants have saved millions of lives of preterm neonates with neonatal Respiratory Distress Syndrome (nRDS). However, a replacement for animal-derived lung surfactants has been sought for decades due to its high manufacturing cost, inaccessibility in low-income countries, and failure to show efficacy when nebulized. This study investigated the use of lipid-coated microbubbles as potential replacements for exogenous lung surfactants. Three different formulations of microbubbles (DPPC with/out PEG40-stearate and poractant alfa) were prepared, and their equilibrium and dynamic surface tensions were tested on a clean air-saline interface or a simulated air-lung fluid interface using a Langmuir-Blodgett trough. In dynamic surface measurements, microbubbles reduced the minimum surface tension compared with the equivalent composition lipid suspension: e.g., PEG-free microbubbles had a minimum surface tension of 4.3 mN/m while the corresponding lipid suspension and poractant alfa had 20.4 ( p ≤ 0.0001) and 21.8 mN/m ( p ≤ 0.0001), respectively. Two potential mechanisms for the reduction of surface tension were found: Fragmentation of the foams created by microbubble coalescence; and clustering of microbubbles in the aqueous subphase disrupting the interfacial phospholipid monolayer. The predominant mechanism appears to depend on the formulation and/or the environment. The use of microbubbles as a replacement for exogenous lung surfactant products thus shows promise and further work is needed to evaluate efficacy in vivo.

Objective: Perfluorocarbon nanodroplets (NDs) have been widely investigated as both diagnostic and therapeutic agents. There remains, however, a challenge in generating NDs that do not vaporize spontaneously but can be activated at ultrasound pressures that do not produce unwanted bioeffects. In previous work, it has been shown that phospholipid-coated perfluorobutane (PFB) NDs can potentially overcome this challenge. The aim of this study was to investigate whether these NDs can promote drug delivery., Methods: A combination of high-speed optical imaging and passive cavitation detection was used to study the acoustic properties of the PFB-NDs in a tissue mimicking phantom. PFB-NDs were exposed to ultrasound at frequencies from 0.5 to 1.5 MHz and peak negative pressures from 0.5 to 3.5 MPa. In addition, the penetration depth of two model drugs (Nile Red and 200 nm diameter fluorescent polymer spheres) into the phantom was measured., Results: PFB NDs were found to be stable in aqueous suspension at both 4°C and 37°C; their size remaining unchanged at 215 ± 11 nm over 24 h. Penetration of both model drugs in the phantom was found to increase with increasing ultrasound peak negative pressure and decreasing frequency and was found to be positively correlated with the energy of acoustic emissions. Extravasation depths >1 mm were observed at 0.5 MHz with pressures <1 MPa., Conclusion: The results of the study thus suggest that PFB NDs can be used both as drug carriers and as nuclei for cavitation to enhance drug delivery without the need for high intensity ultrasound., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetically encodable gas filled particles known as gas vesicles (GVs) have shown promise as a biomolecular contrast agent for ultrasound imaging and have the potential to be used as cavitation nuclei for ultrasound therapy. In this study, we used passive acoustic mapping techniques to characterize GV-seeded cavitation, utilizing 0.5 and 1.6 MHz ultrasound over peak rarefactional pressures ranging from 100 to 2200 kPa. We found that GVs produce cavitation for the duration of the first applied pulse, up to at least 5000 cycles, but that bubble activity diminishes rapidly over subsequent pulses. At 0.5 MHz the frequency content of cavitation emissions was predominantly broadband in nature, whilst at 1.6 MHz narrowband content at harmonics of the main excitation frequency dominated. Simulations and high-speed camera imaging suggest that the received cavitation emissions come not from individual GVs but instead from the coalescence of GV-released gas into larger bubbles during the applied ultrasound pulse. These results will aid the future development of GVs as cavitation nuclei in ultrasound therapy.

Background: People with multiple sclerosis (MS) present varying degrees of disability throughout their disease course. The Multiple Sclerosis Severity Score (MSSS) and the Age-Related Multiple Sclerosis Severity Score (ARMSSS) adjust the Expanded Disability Status Scale (EDSS) according to disease duration and age, respectively. These measures could be useful for quantifying MS severity and as health outcome indicators for benchmarking in population-based settings. The aim of this study was to describe the severity of MS in our health district using the MSSS and ARMSSS and to assess their consistency over time., Methods: This population-based study included patients from our health district who were diagnosed with MS according to the 2010 McDonald criteria, had a disease duration of at least one year and were followed up in our MS unit. Sex, age at onset, disease duration, clinical course, age and irreversible EDSS at the last follow-up visit were collected, and the MSSS and ARMSSS were calculated at two time points: 2017 and 2020., Results: One hundred seventy-seven patients were included in 2017, and 208 in 2020. The prevalence of MS was 90 and 104 per 100,000 inhabitants, respectively. The median MSSS was 1.77 (IQR 0.76-4.28) in 2017 and 2.03 (IQR 0.82-4.36) in 2020. The median ARMSSS was 2.90 (IQR 1.47-5.72) in 2017 and 2.93 (IQR 1.51-5.56) in 2020. No significant differences were found., Conclusions: According to the MSSS and ARMSSS, the severity of MS in our area is mild, and these instruments are consistent. These measures could be reliable health outcome measures., (© 2024. The Author(s).)

Objective: The aim of this study is to demonstrate 3-dimensional (3D) acoustic wave sparsely activated localization microscopy (AWSALM) of microvascular flow in vivo using phase change contrast agents (PCCAs)., Materials and Methods: Three-dimensional AWSALM using acoustically activable PCCAs was evaluated on a crossed tube microflow phantom, the kidney of New Zealand White rabbits, and the brain of C57BL/6J mice through intact skull. A mixture of C 3 F 8 and C 4 F 10 low-boiling-point fluorocarbon gas was used to generate PCCAs with an appropriate activation pressure. A multiplexed 8-MHz matrix array connected to a 256-channel ultrasound research platform was used for transmitting activation and imaging ultrasound pulses and recording echoes. The in vitro and in vivo echo data were subsequently beamformed and processed using a set of customized algorithms for generating 3D super-resolution ultrasound images through localizing and tracking activated contrast agents., Results: With 3D AWSALM, the acoustic activation of PCCAs can be controlled both spatially and temporally, enabling contrast on demand and capable of revealing 3D microvascular connectivity. The spatial resolution of the 3D AWSALM images measured using Fourier shell correlation is 64 μm, presenting a 9-time improvement compared with the point spread function and 1.5 times compared with half the wavelength. Compared with the microbubble-based approach, more signals were localized in the microvasculature at similar concentrations while retaining sparsity and longer tracks in larger vessels. Transcranial imaging was demonstrated as a proof of principle of PCCA activation in the mouse brain with 3D AWSALM., Conclusions: Three-dimensional AWSALM generates volumetric ultrasound super-resolution microvascular images in vivo with spatiotemporal selectivity and enhanced microvascular penetration., Competing Interests: Conflicts of interest and sources of funding: This work was funded in part by the Engineering and Physical Sciences Research Council (EPSRC) under grant EP/T008970/1, EP/T008067/1, and EP/N026942/1; the EPSRC Impact Acceleration Account under grant EP/R511547/1; the National Institute for Health Research i4i under grant NIHR200972; and the British Heart Foundation under grant PG/18/48/33832. The authors report no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background and Objectives: Serum biomarkers are emerging as useful prognostic tools for multiple sclerosis (MS); however, long-term studies are lacking. We aimed to evaluate the long-term prognostic value of the serum levels of neurofilament light chain (NfL), total tau, glial fibrillary acidic protein (GFAP), and chitinase 3-like-1 (CHI3L1) measured close to the time of MS onset., Methods: In this retrospective, exploratory, observational, case and controls study, patients with relapsing-remitting MS (RRMS) with available baseline serum samples and prospectively follow-up in our MS unit for a long time were selected based on their clinical evolution to form two groups: (1) a benign RRMS (bRRMS) group, defined as patients with an Expanded Disability Status Scale (EDSS) score of ≤ 3 at ≥ 10 years of follow-up; (2) an aggressive RRMS (aRRMS) group, defined as patients with an EDSS score of ≥ 6 at ≤ 15 years of follow-up. An age-matched healthy control (HC) group was selected. NfL, total tau, and GFAP serum levels were quantified using a single-molecule array (SIMOA), and CHI3L1 was quantified using ELISA., Results: Thirty-one patients with bRRMS, 19 with aRRMS, and 10 HC were included. The median follow-up time from sample collection was 17.74 years (interquartile range, 14.60-20.37). Bivariate and multivariate analyses revealed significantly higher NfL and GFAP levels in the aRRMS group than in the bRRMS group. A receiver operating characteristic curve analysis identified serum NfL level as the most efficient marker for distinguishing aRRMS from bRRMS., Discussion: This proof-of-concept study comparing benign and aggressive RRMS groups reinforces the potential role of baseline NfL serum levels as a promising long-term disability prognostic marker. In contrast, serum GFAP, total tau, and CHI3L1 levels demonstrated a lower or no ability to differentiate between the long-term outcomes of RRMS., (© 2023. The Author(s).)

Introduction: The role of the kappa-free light chain (kFLC) in the diagnosis of multiple sclerosis (MS) and, to a lesser extent, its role as a medium-term prognostic marker have been extensively studied. This study aimed to explore its potential as a long-term prognostic marker for MS., Methods: We performed an exploratory retrospective observational study by selecting patients systemically followed up in our MS unit with available cerebrospinal fluid and serum samples at the time of initial evaluation. Two groups were defined: benign MS (bMS), defined as patients with Expanded Disability Status Scale (EDSS) ≤ 3 at 10 years of follow-up, and aggressive MS (aMS), defined as patients with EDSS ≥ 6 at 15 years of follow-up. Clinical variables were collected, and the immunoglobulin G (IgG) index, kFLC index, and oligoclonal bands (OCB) were determined for all patients and compared between the groups., Results: Twenty bMS and 15 aMS patients were included in this study. Sixty percent (21/35) were female, and the mean age at the time of the first symptom was 31.5 ± 9.45 years, with no statistical differences between groups. Median follow-up time was 19.8 years (Interquartile range, IQR 15.9-24.6). The median EDSS scores at the last follow-up were 1.5 and 7.5 in the bMS and the aMS group, respectively. No statistically significant differences were found in the kFLC index between the two groups (136.6 vs. 140.27, p=0.59). The IgG index was positive in 62.9% of patients (55% bMS vs. 73.3% aMS, p>0.05), and OCB was positive in 88.6% (90% bMS vs. 86.7% aMS, p>0.05). A significant positive correlation was found between IgG and kFLC indices (r s = 0.85, p<0.001)., Conclusion: Given the absence of differences between the two groups with opposite disease courses, it is unlikely that the kFLC index is a reliable and powerful marker of long-term prognosis in MS., Competing Interests: PA-P, LB, EM, IL, AM-V, LR-P, AM-Y and SM-Y received honoraria for participating on advisory boards and for collaborations as consultants and scientific communications; they also received research support as well as funding for travel and congress expenses from Roche, Biogen Idec, Novartis, TEVA, Merck, Genzyme, Sanofi, Bayer, Almirall, and Celgene. LG-S and VM received funding for travel and congress expenses from The Binding Site. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Arroyo-Pereiro, García-Serrano, Morandeira, Urban, Mas, Framil, León, Muñoz-Vendrell, Matas, Romero-Pinel, Martínez-Yélamos, Martínez-Yélamos and Bau.)

Background: Natalizumab (NTZ) and ocrelizumab (OCR) can be used for the treatment of relapsing-remitting multiple sclerosis (RRMS). In patients treated with NTZ, screening for JC virus (JCV) is mandatory, and a positive serology usually requires a change in treatment after 2 years. In this study, JCV serology was used as a natural experiment to pseudo-randomize patients into NTZ continuation or OCR., Methods: An observational analysis of patients who had received NTZ for at least 2 years and were either changed to OCR or maintained on NTZ, depending on JCV serology status, was performed. A stratification moment (STRm) was established when patients were pseudo-randomized to either arm (NTZ continuation if JCV negativity, or change to OCR if JCV positivity). Primary endpoints include time to first relapse and presence of relapses after STRm and OCR initiation. Secondary endpoints include clinical and radiological outcomes after 1 year., Results: Of the 67 patients included, 40 continued on NTZ (60%) and 27 were changed to OCR (40%). Baseline characteristics were similar. Time to first relapse was not significantly different. Ten patients in the JCV + OCR arm presented a relapse after STRm (37%), four during the washout period, and 13 patients in the JCV-NTZ arm (32.5%, p = 0.701). No differences in secondary endpoints were detected in the first year after STRm., Conclusions: The JCV status can be used as a natural experiment to compare treatment arms with a low selection bias. In our study, switching to OCR versus NTZ continuation led to similar disease activity outcomes., (© 2023. The Author(s).)

Introduction: Prior studies have suggested that cardiovascular risk factors (CVRFs) can affect the prognosis of multiple sclerosis (MS). The aim of this study was to assess if CVRFs affect the early course of MS., Methods: A retrospective observational study was performed, including patients diagnosed with relapsing-remitting MS (RRMS) from 2010 to 2020, with at least 2 years of disease and 6 months follow-up. Age at onset, disease duration, number of relapses, time to confirmed Expanded Disability Status Scale (EDSS) 3.0 and 6.0, and time to secondary progressive MS (SPMS) were collected. Presence and date at onset of hypertension (HT), diabetes mellitus (DM), high low-density lipoprotein cholesterol (LDLc), and smoking during the study period were collected. The primary objective was to assess if CVRFs at the onset of MS are associated with lower time to EDSS 3.0, time to EDSS 6.0, and time to SPMS, using bivariate and multivariate analysis., Results: 281 RRMS patients were included; median age at onset was 33 (IQR 26-39); 69.4% were female. Median EDSS at onset was 1.5 (IQR 1-2.5). Nine patients reached SPMS; 24 patients were diagnosed with HT, 9 with DM, 109 with high LDLc, and 123 were smokers during follow-up. No statistically significant association was found between the presence of CVRF at MS onset and the mentioned clinical outcomes during the MS course., Conclusion: No association was found between CVRFs and the early course of MS in our cohort., (© 2022 S. Karger AG, Basel.)

Background: Patients with relapsing-remitting multiple sclerosis (RRMS) most commonly experience their first symptoms between 20 and 40 years of age. The objective of this study was to investigate how the age at which the first symptoms of RRMS occur has changed over the past decades., Methods: Patients who were followed up in our unit after an initial diagnosis of RRMS using the Poser or McDonald criteria and who experienced their first symptoms between January 1970 and December 2019 were included in the study. The cohort was divided into five groups according to the decade in which the first symptoms appeared. The age at disease onset was compared across decades. Changes in age were also determined after excluding patients with early-onset disease (<18 years of age) and those with late-onset disease (>50 years of age) to avoid bias., Results: The cohort included 1,622 patients with RRMS, 67.6% of whom were women. Among them, 5.9% and 4% had early-onset and late-onset disease, respectively. The mean age ± standard deviation at onset was 31.11 ± 9.82 years, with no differences between men and women. The mean ages at onset were 23.79 ± 10.19 years between 1970 and 1979, 27.86 ± 9.22 years between 1980 and 1989, 30.07 ± 9.32 years between 1990 and 1999, 32.12 ± 9.47 between 2000 and 2009, and 34.28 ± 9.83 years between 2010 and 2019. The ages at disease onset were progressively higher in the later decades; this trend was statistically significant (p < 0.001), with a Pearson linear correlation coefficient R of 0.264 and R 2 of 0.070 (p < 0.001). The results were similar when analysing men and women separately. We conducted an analysis of 1,460 patients (mean age at onset: 31.10 ± 7.99 years), after excluding patients with early-onset and late-onset disease. In this specific subgroup, the mean ages at disease onset were 28.38 ± 8.17 years between 1970 and 1979, 29.22 ± 7.51 years between 1980 and 1989, 30.06 ± 8.02 years between 1990 and 1999, 31.46 ± 7.77 years between 2000 and 2009, and 33.37 ± 7.97 years between 2010 and 2019. The trend was also statistically significant (p < 0.001), with a Pearson linear correlation coefficient R of 0.193 and R 2 of 0.037 (p < 0.001)., Conclusion: Our data showed that the age at RRMS onset has increased over the past decades., Competing Interests: Declaration of Competing Interest Lucía Romero-Pinel, Laura Bau, Elisabet Matas, Isabel León, Albert Muñoz-Vendrell, Pablo Arroyo, Antonio Martínez-Yélamos, and Sergio Martínez-Yélamos received honoraria for participating on advisory boards and for collaborations as consultants and scientific communications; they also received research support as well as funding for travel and congress-attending expenses from Roche, Biogen Idec, Novartis, TEVA, Merck, Genzyme, Sanofi, Bayer, Almirall, and Celgene. Cristina Masuet-Aumatell received honoraria for participating on advisory boards and for collaborations as a consultant and scientific communications and has received research support as well as funding for travel and congress-related expenses from GlaxoSmithKline, Pfizer, Seqirus, Emergent and Sanofi Pasteur., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The efficacy and safety of disease-modifying therapies (DMTs) in multiple sclerosis (MS) are well known; however, owing to their high costs, determining real-world outcomes is essential to evaluate the cost-effectiveness of different therapeutic strategies. This study aimed to investigate the variability in the annual cost of DMTs associated with a relapse-free patient in a representative population cohort of relapsing-remitting MS (RRMS), and whether this could serve as an appropriate health indicator., Methods: We analyzed the patients followed up in our MS clinic during the years 2016 and 2019, and selected patients belonging to our health district diagnosed with RRMS. The treatment cost associated with a relapse-free patient was the ratio between the total cost of DMTs and the number of relapse-free patients, treated and not treated, during the year of the study., Results: A total of 158 patients with RRMS in 2016 and 183 in 2019 were included in our study. In 2016, 101 patients with RRMS (63.9%) received treatment with DMTs and 120 patients (75.9%) remained relapse-free. The mean cost of DMTs per patient in 2016 was €7414.3 (95% confidence interval [CI]: 6325.2-8503.4) considering all the patients (treated and not treated). In 2019, 126 patients (68.9%) received DMTs and 151 patients (82.5%) remained relapse-free. The mean cost of DMTs per patient in 2019 was €6985.4 (95% CI: 5986.9-7983.9) considering all the patients. The cost per year of DMTs to achieve a relapse-free patient was €9762.2 in 2016 and €8465.8 in 2019., Conclusions: The treatment cost per year to achieve a relapse-free patient was stable during successive measurements in the same population. Therefore, it may be considered a good real-world health indicator for patients with RRMS treated with DMTs., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Lucía Romero-Pinel, Laura Bau, Elisabet Matas, Sergio Martínez-Yélamos and Antonio Martínez Yélamos received honoraria compensation to participate in advisory boards, collaborations as a consultant and scientific communications and received research support, funding for travel and congress expenses from Biogen Idec, Novartis, TEVA, MerckSerono, Genzyme, Almirall, Bayer and Roche. Isabel León, Roser Juvany and Ramon Jódar declare nothing to disclose.

Treatment options for patients with pancreatic cancer are limited and survival prospects have barely changed over the past 4 decades. Chemoradiation treatment (CRT) has been used as neoadjuvant therapy in patients with borderline resectable disease to reduce tumour burden and increase the proportion of patients eligible for surgery. Antimetabolite drugs such as gemcitabine and 5-fluorouracil are known to sensitise pancreatic tumours to radiation treatment. Likewise, photodynamic therapy (PDT) has also been shown to enhance the effect of radiation therapy. However, PDT is limited to treating superficial lesions due to the attenuation of light by tissue. The ability of the related technique, sonodynamic therapy (SDT), to enhance CRT was investigated in two murine models of pancreatic cancer (PSN-1 and BxPC-3) in this study. SDT uses low intensity ultrasound to activate an otherwise non-toxic sensitiser, generating toxic levels of reactive oxygen species (ROS) locally. It is applicable to greater target depths than PDT due to the ability of ultrasound to propagate further than light in tissue. Both CRT and the combination of CRT plus SDT delayed tumour growth in the two tumour models. In the PSN-1 model, but not the BxPC-3 model, the combination treatment caused an increase in survival relative to CRT alone (p = 0.038). The improvement in survival conferred by the addition of SDT in this model may be related to differences in tumour architecture between the two models. MRI and US images showed that PSN-1 tumours were less well perfused and vascularised than BxPC-3 tumours. This poor vascularisation may explain why PSN-1 tumours were more susceptible to the effects of vascular damage exerted by SDT treatment., (Copyright © 2021. Published by Elsevier B.V.)

A combination of ultrahigh-speed optical imaging (5 × 10 6 frames/s), B-mode ultrasound and passive cavitation detection was used to study the vaporization process and determine both the acoustic droplet vaporization (ADV) and inertial cavitation (IC) thresholds of phospholipid-coated perfluorobutane nanodroplets (PFB NDs, diameter = 237 ± 16 nm). PFB NDs have not previously been studied with ultrahigh-speed imaging and were observed to form individual microbubbles (1-10 μm) within two to three cycles and subsequently larger bubble clusters (10-50 μm). The ADV and IC thresholds did not statistically significantly differ and decreased with increasing pulse length (20-20,000 cycles), pulse repetition frequency (1-100 Hz), concentration (10 8 -10 10 NDs/mL), temperature (20°C-45°C) and decreasing frequency (1.5-0.5 MHz). Overall, the results indicate that at frequencies of 0.5, 1.0 and 1.5 MHz, PFB NDs can be vaporized at moderate peak negative pressures (<2.0 MPa), pulse lengths and pulse repetition frequencies. This finding is encouraging for the use of PFB NDs as cavitation agents, as these conditions are comparable to those required to achieve therapeutic effects with microbubbles, unlike those reported for higher-boiling-point NDs. The differences between the optically and acoustically determined ADV thresholds, however, suggest that application-specific thresholds should be defined according to the biological/therapeutic effect of interest., Competing Interests: Conflict of interest statement The authors declare no conflicts of interest., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis., Competing Interests: conflict of interest statement John Callan, Anthony McHale and Eleanor Stride are co-founders of a spin out company, SonoTarg Ltd. and inventors on a patent (US20180344872A1) which covers one of the formulations reported in this paper. There was, however, no involvement of SonoTarg in the design, conduct or funding of this study and SonoTarg is not currently pursuing applications in chemoradiation therapy., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Purpose: Chemoradiation therapy is the standard of care in muscle-invasive bladder cancer (MIBC). Although agents such as gemcitabine can enhance tumor radiosensitivity, their side effects can limit patient eligibility and treatment efficacy. This study investigates ultrasound and microbubbles for targeting gemcitabine delivery to reduce normal-tissue toxicity in a murine orthotopic MIBC model., Materials and Methods: CD1-nude mice were injected orthotopically with RT112 bladder tumor cells. Conventional chemoradiation involved injecting gemcitabine (10 mg/kg) before 6 Gy targeted irradiation of the bladder area using the Small Animal Radiation Research Platform (SARRP). Ultrasound-mediated gemcitabine delivery (10 mg/kg gemcitabine) involved either coadministration of microbubbles with gemcitabine or conjugating gemcitabine onto microbubbles followed by exposure to ultrasound (1.1 MHz center frequency, 1 MPa peak negative pressure, 1% duty cycle, and 0.5 Hz pulse repetition frequency) before SARRP irradiation. The effect of ultrasound and microbubbles alone was also tested. Tumor volumes were measured by 3D ultrasound imaging. Acute normal-tissue toxicity from 12 Gy to the lower bowel area was assessed using an intestinal crypt assay in mice culled 3.75 days posttreatment., Results: A significant delay in tumor growth was observed with conventional chemoradiation therapy and both microbubble groups (P < .05 compared with the radiation-only group). Transient weight loss was seen in the microbubble groups, which resolved within 10 days posttreatment. A positive correlation was found between weight loss on day 3 posttreatment and tumor growth delay (P < .05; R 2 = 0.76). In contrast with conventional chemoradiation therapy, ultrasound-mediated drug delivery methods did not exacerbate the acute intestinal toxicity using the crypt assay., Conclusions: Ultrasound and microbubbles offer a promising new approach for improving chemoradiation therapy for muscle-invasive bladder cancer, maintaining a delay in tumor growth but with reduced acute intestinal toxicity compared with conventional chemoradiation therapy., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vaccinia virus (VV) is a powerful tool for cancer treatment with the potential for tumor tropism, efficient cell-to-cell spread, rapid replication in cancer cells, and stimulation of anti-tumor immunity. It has a well-defined safety profile and is being assessed in late-stage clinical trials. However, VV clinical utility is limited by rapid bloodstream neutralization and poor penetration into tumors. These factors have often restricted its route of delivery to intratumoral or intrahepatic artery injection and may impede repeat dosing. Chemical stealthing improves the pharmacokinetics of non-enveloped viruses, but it has not yet been applied to enveloped viruses such as VV. In the present study, amphiphilic polymer was used to coat VV, leading to reduced binding of a neutralizing anti-VV antibody (81.8% of polymer-coated VV [PCVV] staining positive versus 97.1% of VV [p = 0.0038]). Attachment of anti-mucin-1 (aMUC1) targeting antibody, to give aMUC1-PCVV, enabled binding of the construct to MUC1. In high MUC1 expressing CAPAN-2 cells, infection with PCVV was reduced compared to VV, while infection was restored with aMUC1-PCVV. Pharmacokinetics of aMUC1-PCVV, PCVV, and VV were evaluated. After intravenous (i.v.) injection of 1 × 10 8 viral genomes (VG) or 5 × 10 8 VG, circulation time for PCVV and aMUC1-PCVV was increased, with ~5-fold higher circulating dose at 5 min versus VV., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Aims: Testing ultrasound-mediated cavitation for enhanced delivery of the therapeutic antibody cetuximab to tumors in a mouse model. Methods: Tumors with strong EGF receptor expression were grown bilaterally. Cetuximab was coadministered intravenously with cavitation nuclei, consisting of either the ultrasound contrast agent Sonovue or gas-stabilizing nanoscale SonoTran Particles. One of the two tumors was exposed to focused ultrasound. Passive acoustic mapping localized and monitored cavitation activity. Both tumors were then excised and cetuximab concentration was quantified. Results: Cavitation increased tumoral cetuximab concentration. When nucleated by Sonovue, a 2.1-fold increase (95% CI 1.3- to 3.4-fold) was measured, whereas SonoTran Particles gave a 3.6-fold increase (95% CI 2.3- to 5.8-fold). Conclusions: Ultrasound-mediated cavitation, especially when nucleated by nanoscale gas-entrapping particles, can noninvasively increase site-specific delivery of therapeutic antibodies to solid tumors.

Recent pre-clinical studies have demonstrated the potential of combining chemotherapy and sonodynamic therapy for the treatment of pancreatic cancer. Oxygen-loaded magnetic microbubbles have been explored as a targeted delivery vehicle for this application. Despite preliminary positive results, a previous study identified a significant practical challenge regarding the co-alignment of the magnetic and ultrasound fields. The aim of this study was to determine whether this challenge could be addressed through the use of a magnetic-acoustic device (MAD) combining a magnetic array and ultrasound transducer in a single unit, to simultaneously concentrate and activate the microbubbles at the target site. in vitro experiments were performed in tissue phantoms and followed by in vivo treatment of xenograft pancreatic cancer (BxPC-3) tumours in a murine model. In vitro, a 1.4-fold (p < .01) increase in the deposition of a model therapeutic payload within the phantom was achieved using the MAD compared to separate magnetic and ultrasound devices. In vivo, tumours treated with the MAD had a 9% smaller mean volume 8 days after treatment, while tumours treated with separate devices or microbubbles alone were respectively 45% and 112% larger. This substantial and sustained decrease in tumour volume suggests that the proposed drug delivery approach has the potential to be an effective neoadjuvant therapy for pancreatic cancer patients., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The optimal clinical exploitation of viruses as gene therapy or oncolytic vectors will require them to be administered intravenously. Strategies must therefore be deployed to enable viruses to survive the harsh neutralizing environment of the bloodstream and achieve deposition within and throughout target tissues or tumor deposits. This chapter describes the genetic and chemical engineering approaches that are being developed to overcome these challenges.

Ultrasound-induced cavitation has been proposed as a strategy to tackle the challenge of inadequate extravasation, penetration and distribution of therapeutics into tumours. Here, the ability of microbubbles, droplets and solid gas-trapping particles to facilitate mass transport and extravasation of a model therapeutic agent following ultrasound-induced cavitation is investigated. Significant extravasation and penetration depths on the order of millimetres are achieved with all three agents, including the range of pressures and frequencies achievable with existing clinical ultrasound systems. Deeper but highly directional extravasation was achieved with frequencies of 1.6 and 3.3 MHz compared with 0.5 MHz. Increased extravasation was observed with increasing pulse length and exposure time, while an inverse relationship is observed with pulse repetition frequency. No significant cell death or any haemolytic activity in human blood was observed at clinically relevant concentrations for any of the agents. Overall, solid gas-trapping nanoparticles were found to enable the most extensive extravasation for the lowest input acoustic energy, followed by microbubbles and then droplets. The ability of these agents to produce sustained inertial cavitation activity whilst being small enough to follow the drug out of the circulation and into diseased tissue, combined with a good safety profile and the possibility of real-time monitoring, offers considerable potential for enhanced drug delivery of unmodified drugs in oncological and other biomedical applications., (Copyright © 2018. Published by Elsevier Inc.)

The advancement of ultrasound-mediated therapy has stimulated the development of drug-loaded microbubble agents that can be targeted to a region of interest through an applied magnetic field prior to ultrasound activation. However, the need to incorporate therapeutic molecules while optimizing the responsiveness to both magnetic and acoustic fields and maintaining adequate stability poses a considerable challenge for microbubble synthesis. The aim of this study was to evaluate three different methods for incorporating iron oxide nanoparticles (IONPs) into phospholipid-coated microbubbles using (1) hydrophobic IONPs within an oil layer below the microbubble shell, (2) phospholipid-stabilized IONPs within the shell, or (3) hydrophilic IONPs noncovalently bound to the surface of the microbubble. All microbubbles exhibited similar acoustic response at both 1 and 7 MHz. The half-life of the microbubbles was more than doubled by the addition of IONPs by using both surface and phospholipid-mediated loading methods, provided the lipid used to coat the IONPs was the same as that constituting the microbubble shell. The highest loading of IONPs per microbubble was also achieved with the surface loading method, and these microbubbles were the most responsive to an applied magnetic field, showing a 3-fold increase in the number of retained microbubbles compared to other groups. For the purpose of drug delivery, surface loading of IONPs could restrict the attachment of hydrophilic drugs to the microbubble shell, but hydrophobic drugs could still be incorporated. In contrast, although the incorporation of phospholipid IONPs produced more weakly magnetic microbubbles, it would not interfere with hydrophilic drug loading on the surface of the microbubble.

The efficient penetration of drugs into tumors is a major challenge that remains unmet. Reported herein is a strategy to promote extravasation and enhanced penetration using inertial cavitation initiated by focused ultrasound and cone-shaped gold nanoparticles that entrap gas nanobubbles. The cones are capable of initiating inertial cavitation under pressures and frequencies achievable with existing clinical ultrasound systems and of promoting extravasation and delivery of a model large therapeutic molecule in an in vitro tissue mimicking flow phantom, achieving penetration depths in excess of 2 mm. Ease of functionalization and intrinsic imaging capabilities provide gold with significant advantages as a material for biomedical applications. The cones show neither cytotoxicity in Michigan Cancer Foundation (MCF)-7 cells nor hemolytic activity in human blood at clinically relevant concentrations and are found to be colloidally stable for at least 5 d at 37 °C and several months at 4 °C., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Giant unilamellar vesicles (GUVs) are well-established model systems for studying membrane structure and dynamics. Electroformation, also referred to as electroswelling, is one of the most prevalent methods for producing GUVs, as it enables modulation of the lipid hydration process to form relatively monodisperse, defect-free vesicles. Currently, however, it is expensive and time-consuming compared with other methods. In this study, we demonstrate that 1,2-dioleoyl- sn -glycero-3-phosphatidylcholine GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes, such as commercially available syringe needles, without any evidence of lipid oxidation or hydrolysis.

In the present study, CSF concentrations of NFL, t-tau, p-tau, GFAP, S-100B, YKL-40, MCP-1, α-sAPP, β-sAPP, and Aβ38, Aβ40, Aβ42 were measured in 324 MS patients to test whether a correlation among the biomarkers exists and whether the profile of CSF biomarkers varies among the different types of MS. The CSF concentrations of NFL were significantly higher in RRMS while CSF concentrations of GFAP were higher in PPMS. CSF concentrations of NFL correlated with YKL-40 in CIS patients while CSF concentrations of GFAP correlated with YKL-40 in RRMS patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cell-adhesion molecules (CAMs) dynamics in Multiple Sclerosis (MS) patients have been widely studied after Natalizumab (NTZ) introduction. However, their temporal dynamics after NTZ withdrawal (NTZ-W) has not been described. We prospectively evaluate changes in the expression levels of CAMs (CD49d, CD29, L-Selectin and CD11a) involved in T cell migration of 22 MS patients after NTZ-W. CD49d, CD29 and CD11a expression experienced a continuous increase expression two months after NTZ-W and Cd49d expression at month six after NTZ-W correlated to NTZ treatment duration, both in CD45 + CD4 + and CD45 + CD8 + . CD49d expression up to month three after NTZ-W was related to MS activity in CD45 + CD8 + at the end of the study. Results from this study suggest that patients with a longer NTZ treatment are more susceptible to present a "molecular rebound" after NTZ-W. CD49d determination may be a useful tool to closely monitor MS activity in patients who interrupt NTZ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study is to investigate whether induction of myxovirus resistance protein A (MxA) mRNA after 3 months of interferon-β administration is related to the treatment response in multiple sclerosis (MS) patients. In this prospective study, MS patients were enrolled before starting treatment. Demographic, clinical and radiological variables were recorded. Blood samples were obtained before, and at 3 and 12 months after interferon-β treatment. Real-time PCR was used to analyze MxA mRNA expression. Patients were classified as MxA-low or -high depending on MxA levels at baseline, and as MxA-induced or -non-induced according to whether an increase in MxA expression was detected at month 3. Time to the next relapse was investigated using Cox proportional hazards regression analysis. One hundred and four patients were selected and followed for a median of 2.2 years (IQR 1.6-3.5). On Cox regression analysis, a higher EDSS score before treatment (HR 1.57; 95 % CI 1.02-2.40; p = 0.039), MxA-high status at baseline (HR 2.71; 95 % CI 1.26-5.81; p = 0.010), and MxA-non-induced at month 3 (HR 2.49; 95 % CI 1.08-5.68; p = 0.031), were predictors of poor response to interferon-β in naïve MS patients. Patients showing a lower capacity for MxA induction following 3 months of interferon-β treatment are more likely to be non-responders to this therapy.

Myxovirus resistance protein A (MxA) is a molecule induced after interferon-β injection. The aim of this study was to investigate whether MxA determination one year after starting interferon-β can predict treatment response in multiple sclerosis patients. MxA mRNA expression was evaluated in blood samples obtained at baseline and at month 12. Clinical variables were prospectively recorded. A threshold of 5 was defined to establish MxA induction. On survival analysis, time to the next relapse and to EDSS progression were significantly longer in patients showing MxA induction, suggesting that MxA induction after one year may be useful to identify interferon-β responders., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To investigate glial and neuronal biomarkers in cerebrospinal fluid (CSF) samples from patients with relapsing-remitting multiple sclerosis (RRMS) and clinically isolated syndrome (CIS) suggestive of multiple sclerosis (MS), and to evaluate their ability to predict conversion from CIS to clinically definite MS (CDMS) and also disability progression in MS., Methods: CSF levels of neurofilament light protein (NFL), t-tau, p-tau, glial fibrillary acidic protein (GFAP), S-100B, human chitinase 3-like 1 protein (YKL-40), monocyte chemoattractant protein-1 (MCP-1), α-sAPP and β-sAPP; and Aβ38, Aβ40 and Aβ42, were analyzed in 109 CIS patients and 192 RRMS patients. The mean follow-up time of these 301 patients was 11.7 ± 6.4 years., Results: High levels of NFL were associated with early conversion from CIS to CDMS (hazard ratio (HR) with 95% confidence interval (CI): 2.69 (1.75 - 4.15); p < 0.0001). High levels of YKL-40 and GFAP were associated with earlier progression in the Expanded Disability Status Scale (EDSS), score 3: YKL-40 (HR (95% CI): 2.78 (1.48 - 5.23); p = 0.001) and GFAP (HR (95% CI): 1.83 (1.01 - 3.35); p = 0.04). High levels of YKL-40 were associated with earlier progression to EDSS 6 (HR (95% CI): 4.57 (1.01 - 20.83); p = 0.05)., Conclusions: CSF levels of NFL in CIS patients are an independent prognostic marker for conversion to CDMS. Whereas, CSF levels of YKL-40 and GFAP are independent prognostic markers for disability progression in MS., (© The Author(s), 2015.)

Introduction: We evaluated the effectiveness of natalizumab in patients with highly active, relapsing-remitting multiple sclerosis (HA-RRMS) to identify baseline predictors associated with freedom from disease activity., Methods: We analyzed 70 patients treated with natalizumab and followed for at least 1 year with progression of disability of ≥1 point on the EDSS before starting therapy. We recorded freedom from clinical activity, radiological activity, and disease activity (clinical and radiological)., Results: The median (IQR) follow-up was 2.3 (2.0-3.8) years. Of the 52 patients who completed 2 years of treatment, 25 were free of disease activity (48.1%). The ARR decreased from a mean ± SD of 2.49 ± 0.86 at baseline to 0.47 ± 0.83 at the end of the first year (p < 0.001) and 0.34 ± 0.69 at the end of the second year (p < 0.001). The percentage of patients with gadolinium-enhanced lesions decreased from 21 at baseline to 5.7 at the end of the first year (p < 0.001) and to 5.8 during the second year (p < 0.005). Baseline EDSS ≤3.0 was significantly associated with freedom from disease activity (OR, 2.49; 95% CI, 1.24-4.99; p = 0.010)., Conclusions: Natalizumab is effective in patients with HA-RRMS. Baseline EDSS ≤3.0 increases the probability of remaining disease-free in HA-RRMS treated with natalizumab., (© 2015 S. Karger AG, Basel.)

Background: Myxovirus resistance protein A (MxA) is a molecule induced after interferon-beta injection, mostly used to evaluate its bioactivity. There is little available data on clinical utility of baseline MxA mRNA status. The objective of the study is to investigate whether baseline MxA mRNA expression can predict relapse and disease progression in multiple sclerosis patients treated with interferon-beta., Methods: Baseline blood samples were obtained before the first interferon-beta dose was administered to evaluate MxA mRNA expression using real-time polymerase chain reaction (PCR). Demographic and clinical variables were prospectively recorded to define treatment responder and non responder groups., Results: 104 patients were included in the study. Baseline MxA mRNA expression was significantly lower in the group of patients who met the definition of responders (1.07 vs 1.95, Student t test, p<0.0001). A threshold of 1.096 was established using Receiver Operating Characteristic analysis to differentiate between responders and non-responders (sensitivity 73.9%, specificity 69.0%). Survival analysis using this threshold showed that time to next relapse (p<0.0001) and to EDSS progression (p = 0.01) were significantly higher in patients with lower MxA titers., Conclusion: The results suggest that baseline MxA mRNA levels may be useful for predicting whether multiple sclerosis patients will respond or not to interferon-beta treatment.