Ataxia telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Human induced pluripotent stem cells generated from epilepsy patients for use as in vitro models for drug screening.

In this paper, we describe the generation and validation of human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) from 6 epilepsy patients using a non-integrative Sendai virus vector. These human cellular models will enable patient-specific drug screening to improve outcomes for individuals with this disorder.

Generation of induced pluripotent stem cell lines from peripheral blood mononuclear cells of three drug resistant and three drug responsive epilepsy patients.

Epilepsy is a common neurological disorder characterized by seizures. Unfortunately, 30-40% of all epilepsy patients are resistant to at least two or more anti-seizure medications. Attempts to treat these patients and prevent further seizures necessitates multiple drug trials for the patient. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) from 3 drug responsive and 3 drug resistant patients, using a non-integrative Sendai virus vector. These lines can be used to generate 2D and 3D patient-specific human cellular models that will enable personalised drug screening and pharmacogenomic studies.

Ataxia Telangiectasia iPSC line generated from a patient olfactory biopsy identifies novel disease-causing mutations.

Ataxia Telangiectasia is a rare autosomal recessive disorder caused by a mutated ATM gene. The most debilitating symptom of Ataxia Telangiectasia is the progressive neurodegeneration of the cerebellum, though the molecular mechanisms driving this degeneration remains unclear. Here we describe the generation and validation of an induced pluripotent stem cell (iPSC) line from an olfactory biopsy from a patient with Ataxia Telangiectasia. Sequencing identified two previously unreported disease-causing mutations in the ATM gene. This line can be used to generate 2D and 3D patient-specific neuronal models enabling investigations into the mechanisms underlying neurodegeneration.

Reprogramming of human olfactory neurosphere-derived cells from olfactory mucosal biopsies of a control cohort.

Human olfactory neurosphere-derived (ONS) cells are derived from the olfactory mucosa and display some progenitor- and neuronal cell-like properties, making them useful models of neurological disorders. However, they lack several important characteristics of true neurons, which can be overcome using induced pluripotent stem cell (iPSC) -derived neurons. Here we describe, for the first time, the generation and validation of an iPSC line from an olfactory biopsy from a control cohort member. This data lays the groundwork for future reprogramming of ONS cells, which can be used to generate neuronal models and compliment current ONS cell-based investigations into numerous neurological disorders.

[Multiple sclerosis treatment consensus group (MSTCG): position paper on disease-modifying treatment of multiple sclerosis 2021 (white paper)]. / Multiple Sklerose Therapie Konsensus Gruppe (MSTKG): Positionspapier zur verlaufsmodifizierenden Therapie der Multiplen Sklerose 2021 (White Paper).

Multiple sclerosis is a complex, autoimmune-mediated disease of the central nervous system characterized by inflammatory demyelination and axonal/neuronal damage. The approval of various disease-modifying therapies and our increased understanding of disease mechanisms and evolution in recent years have significantly changed the prognosis and course of the disease. This update of the Multiple Sclerosis Therapy Consensus Group treatment recommendation focuses on the most important recommendations for disease-modifying therapies of multiple sclerosis in 2021. Our recommendations are based on current scientific evidence and apply to those medications approved in wide parts of Europe, particularly German-speaking countries (Germany, Austria, Switzerland).

"Hitting the spot": Developing individuals with lived-experience of health and social care as facilitators to deliver a course to enhance public involvement in research - a Welsh perspective.

PLAIN ENGLISH SUMMARY: Public involvement in research has become an important and integral part of the research process in health and social care, from the early stages of research prioritisation and development to the later stages of research conduct and dissemination. Learning and development opportunities, including training, can assist the public and researchers in working together in the research process, and a training schedule exists in Wales for this purpose. One of the key components of this training schedule in Wales is the course Involving the Public in the Design and Conduct of Research: Building Research Partnerships. Building on the existing successes of this UK-wide course, first developed by Macmillan Cancer Support, a project was established between Health and Care Research Wales and Macmillan Cancer Support to develop three members of the Involving People Network into trained facilitators. Once trained, the aim was for the three facilitators to deliver the course in Wales. Macmillan Cancer Support and Health and Care Research Wales selected, through a competitive process, three members of the Involving People Network to use their lived experience of Involvement in research projects, as well as any lived experience of a physical or mental health condition or illness, to become facilitators of the course in the unique context of public involvement in research in Wales. Through this process many benefits were realised, including developing the course content and its delivery in Wales, as well as building the skills and confidence of the individuals themselves as facilitators. This has contributed to a continuing commitment to the sustainable delivery of the Involving the Public in the Design and Conduct of Research: Building Research Partnerships course in Wales and a combined approach to addressing any challenges and obstacles which presented. ABSTRACT: Health and Care Research Wales has a strategic aim to Ensure public involvement and engagement is central to what we do and visible in all elements of it. As part of the ongoing development of the Health and Care Research Wales Training Programme a project was initiated to develop members of the public as facilitators to deliver a public involvement in research course. The project was undertaken in collaboration with Macmillan Cancer Support and was advertised via the Involving People Network in Wales. Three trainee facilitators were recruited, from 14 people that applied, to deliver a public involvement in research training course, the Building Research Partnerships course, as it was known then, originally developed for and by Macmillan Cancer Support. As members of the Involving People Network, the trainees were given training, mentorship, financial and administrative support to develop their role as facilitators over a two year period. This has been reciprocated with incredible commitment, ongoing course delivery in Wales, excellent course evaluations, course review and involvement in future planning. Through this project several benefits were realised, including developing the course content and its delivery and building the skills and confidence of the individual facilitators themselves. Additionally, and importantly, the project team found that patients and members of the public who are given appropriate training and support can greatly enhance a research training programme and act as highly effective ambassadors to further the cause of public involvement in research.

Intravenous immune-modifying nanoparticles as a therapy for spinal cord injury in mice.

Intravenously infused synthetic 500nm nanoparticles composed of poly(lactide-co-glycolide) are taken up by blood-borne inflammatory monocytes via a macrophage scavenger receptor (macrophage receptor with collagenous structure), and the monocytes no longer traffic to sites of inflammation. Intravenous administration of the nanoparticles after experimental spinal cord injury in mice safely and selectively limited infiltration of hematogenous monocytes into the injury site. The nanoparticles did not bind to resident microglia, and did not change the number of microglia in the injured spinal cord. Nanoparticle administration reduced M1 macrophage polarization and microglia activation, reduced levels of inflammatory cytokines, and markedly reduced fibrotic scar formation without altering glial scarring. These findings thus implicate early-infiltrating hematogenous monocytes as highly selective contributors to fibrosis that do not play an indispensable role in gliosis after SCI. Further, the nanoparticle treatment reduced accumulation of chondroitin sulfate proteoglycans, increased axon density inside and caudal to the lesion site, and significantly improved functional recovery after both moderate and severe injuries to the spinal cord. These data provide further evidence that hematogenous monocytes contribute to inflammatory damage and fibrotic scar formation after spinal cord injury in mice. Further, since the nanoparticles are simple to administer intravenously, immunologically inert, stable at room temperature, composed of an FDA-approved material, and have no known toxicity, these findings suggest that the nanoparticles potentially offer a practical treatment for human spinal cord injury.

Patient perspectives on wait times and the impact on their life: A waiting room survey in a chronic pain clinic.

BACKGROUND AND AIMS: Chronic pain is a debilitating condition that requires prompt access to care for effective treatment. Wait times for care often exceed benchmark recommendations, with potential consequences to patient health outcomes. The goal of this paper is to gain the perspectives of patients attending a chronic pain clinic regarding the acceptability of current wait times and the impact of their experiences of waiting for chronic pain care. METHODS: The study took place in a chronic pain clinic at an academic-affiliated teaching hospital in Ottawa, Canada, which housed seven clinicians at the time of the study. New patients attending the chronic pain clinic between July 14, 2014 and August 5, 2015 were eligible to participate based on the availability of the research and clerical staff who administered the survey on a variety of days over the course of the study. Patients completed a self-administered 29-item survey. The survey took approximately five to ten minutes to complete. Questions pertained to patients' socioeconomic factors, chronicity and burden of pain symptoms, and satisfaction with current wait times. Actual wait times were self-reported. Survey results were entered into an Excel spreadsheet, exported to SPSS, and coded numerically to facilitate descriptive analyses using comparative graphs and tables. Open-text responses were reviewed by the authors. RESULTS: Sixty-six patients completed the survey. While 83% of patients stated that their ideal wait time was less than three months, 32% reported receiving an appointment within this period, and 31% reported waiting a year or more. Only 37% of patients felt the wait time for their appointment was appropriate. During their wait, 41% of patients reported receiving written information about chronic pain and 47% were referred to a local chronic pain management group. 94% reported interference with social/recreational activities and normal activities of daily living, 31% had to miss work or school due to the frequency of ongoing symptoms, and 22% reported being unable to attend work or school altogether. Furthermore, 37% of patients reported visiting the emergency room within the previous year and 65% worried about having a serious undiagnosed disease. CONCLUSIONS: Our study found that wait times for chronic pain care, even those triaged as urgent cases, far exceeded what patients considered ideal. Only a third of patients received care within three months of making their appointment, while nearly another third waited over a year. During the waiting period, nearly all patients experienced some impact on their day-to-day activities and work or school attendance, half were unemployed, and nearly a quarter reported a complete inability to attend work or school because of pain. IMPLICATIONS: Wait times for chronic pain care exceed timelines deemed acceptable by patients, causing anxiety and reducing function. The patient perspective must be considered in initiatives attempting to improve access to care for this population with specific needs and goals. Innovative solutions, such as electronic consultation and shared care models, hold promise.

Targeting the GM-CSF receptor for the treatment of CNS autoimmunity.

In multiple sclerosis (MS), there is a growing interest in inhibiting the pro-inflammatory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to evaluate the therapeutic potential and underlying mechanisms of GM-CSF receptor alpha (Rα) blockade in animal models of MS. We show that GM-CSF signaling inhibition at peak of chronic experimental autoimmune encephalomyelitis (EAE) results in amelioration of disease progression. Similarly, GM-CSF Rα blockade in relapsing-remitting (RR)-EAE model prevented disease relapses and inhibited T cell responses specific for both the inducing and spread myelin peptides, while reducing activation of mDCs and inflammatory monocytes. In situ immunostaining of lesions from human secondary progressive MS (SPMS), but not primary progressive MS patients shows extensive recruitment of GM-CSF Rα+ myeloid cells. Collectively, this study reveals a pivotal role of GM-CSF in disease relapses and the benefit of GM-CSF Rα blockade as a potential novel therapeutic approach for treatment of RRMS and SPMS.

Biodegradable antigen-associated PLG nanoparticles tolerize Th2-mediated allergic airway inflammation pre- and postsensitization.

Specific immunotherapy (SIT) is the most widely used treatment for allergic diseases that directly targets the T helper 2 (Th2) bias underlying allergy. However, the most widespread clinical applications of SIT require a long period of dose escalation with soluble antigen (Ag) and carry a significant risk of adverse reactions, particularly in highly sensitized patients who stand to benefit most from a curative treatment. Thus, the development of safer, more efficient methods to induce Ag-specific immune tolerance is critical to advancing allergy treatment. We hypothesized that antigen-associated nanoparticles (Ag-NPs), which we have used to prevent and treat Th1/Th17-mediated autoimmune disease, would also be effective for the induction of tolerance in a murine model of Th2-mediated ovalbumin/alum-induced allergic airway inflammation. We demonstrate here that antigen-conjugated polystyrene (Ag-PS) NPs, although effective for the prophylactic induction of tolerance, induce anaphylaxis in presensitized mice. Antigen-conjugated NPs made of biodegradable poly(lactide-co-glycolide) (Ag-PLG) are similarly effective prophylactically, are well tolerated by sensitized animals, but only partially inhibit Th2 responses when administered therapeutically. PLG NPs containing encapsulated antigen [PLG(Ag)], however, were well tolerated and effectively inhibited Th2 responses and airway inflammation both prophylactically and therapeutically. Thus, we illustrate progression toward PLG(Ag) as a biodegradable Ag carrier platform for the safe and effective inhibition of allergic airway inflammation without the need for nonspecific immunosuppression in animals with established Th2 sensitization.

Targeted immunomodulation using antigen-conjugated nanoparticles.

The growing prevalence of nanotechnology in the fields of biology, medicine, and the pharmaceutical industry is confounded by the relatively small amount of data on the impact of these materials on the immune system. In addition to concerns surrounding the potential toxicity of nanoparticle (NP)-based delivery systems, there is also a demand for a better understanding of the mechanisms governing interactions of NPs with the immune system. Nanoparticles can be tailored to suppress, enhance, or subvert recognition by the immune system. This 'targeted immunomodulation' can be achieved by delivery of unmodified particles, or by modifying particles to deliver drugs, proteins/peptides, or genes to a specific site. In order to elicit the desired, beneficial immune response, considerations should be made at every step of the design process: the NP platform itself, ligands, and other modifiers, the delivery route, and the immune cells that will encounter the conjugated NPs can all impact host immune responses.

A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease.

Targeted immune tolerance is a coveted therapy for the treatment of a variety of autoimmune diseases, as current treatment options often involve nonspecific immunosuppression. Intravenous (iv) infusion of apoptotic syngeneic splenocytes linked with peptide or protein autoantigens using ethylene carbodiimide (ECDI) has been demonstrated to be an effective method for inducing peripheral, antigen-specific tolerance for treatment of autoimmune disease. Here, we show the ability of biodegradable poly(lactic-co-glycolic acid) (PLG) nanoparticles to function as a safe, cost-effective, and highly efficient alternative to cellular carriers for the induction of antigen-specific T cell tolerance. We describe the formulation of tolerogenic PLG particles and demonstrate that administration of myelin antigen-coupled particles both prevented and treated relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a CD4 T cell-mediated mouse model of multiple sclerosis (MS). PLG particles made on-site with surfactant modifications surpass the efficacy of commercially available particles in their ability to couple peptide and to prevent disease induction. Most importantly, myelin antigen-coupled PLG nanoparticles are able to significantly ameliorate ongoing disease and subsequent relapses when administered at onset or at peak of acute disease, and minimize epitope spreading when administered during disease remission. Therapeutic treatment results in significantly reduced CNS infiltration of encephalitogenic Th1 (IFN-γ) and Th17 (IL-17a) cells as well as inflammatory monocytes/macrophages. Together, these data describe a platform for antigen display that is safe, low-cost, and highly effective at inducing antigen-specific T cell tolerance. The development of such a platform carries broad implications for the treatment of a variety of immune-mediated diseases.

A vaccine against CCR5 protects a subset of macaques upon intravaginal challenge with simian immunodeficiency virus SIVmac251.

As an alternative to targeting human immunodeficiency virus (HIV), we have developed vaccines targeting CCR5, a self-protein critically involved in HIV replication and pathogenesis. By displaying peptides derived from CCR5 at high density on the surface of virus-like particles, we can efficiently induce high-titer IgG antibodies against this self-molecule. Here, we investigated whether prophylactic immunization of rhesus macaques with a particle-based vaccine targeting two regions of macaque CCR5 could prevent or suppress vaginal infection with highly virulent SIVmac251. Twelve macaques were vaccinated with a bacteriophage Qß-based vaccine targeting macaque CCR5 (Qß.CCR5). Six control animals were immunized with the Qß platform alone. All animals immunized with Qß.CCR5 developed high-titer anti-CCR5 antibody responses. Macaques were vaginally challenged with a high dose of SIVmac251. The mean peak viral RNA levels in the vaccinated groups were 30-fold lower than in the control group (10(6.8) versus 10(8.3) copies/ml plasma). Three of the 12 vaccinated macaques dramatically suppressed simian immunodeficiency virus (SIV) replication: peak viral loads were low (10(3) to 10(4) RNA copies/ml), and SIV RNA became undetectable from 6 weeks onward. No viral RNA or DNA could be detected in colon and lymph node biopsy specimens collected 13 months after challenge. In vivo depletion of CD8(+) cells failed to induce a viral rebound. However, once anti-CCR5 antibody responses had waned, the 3 animals became infected after intravaginal and/or intravenous rechallenge. In conclusion, vaccination against CCR5 was associated with dramatic suppression of virus replication in a subset (25%) of macaques. These data support further research of vaccination against CCR5 to combat HIV infection.

Optical tomographic imaging of near infrared imaging agents quantifies disease severity and immunomodulation of experimental autoimmune encephalomyelitis in vivo.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model that captures many of the hallmarks of human multiple sclerosis (MS), including blood-brain barrier (BBB) breakdown, inflammation, demyelination and axonal destruction. The standard clinical score measurement of disease severity and progression assesses functional changes in animal mobility; however, it does not offer information regarding the underlying pathophysiology of the disease in real time. The purpose of this study was to apply a novel optical imaging technique that offers the advantage of rapid imaging of relevant biomarkers in live animals. METHODS: Advances in non-invasive fluorescence molecular tomographic (FMT) imaging, in combination with a variety of biological imaging agents, offer a unique, sensitive and quantifiable approach to assessing disease biology in living animals. Using vascular (AngioSense 750EX) and protease-activatable cathepsin B (Cat B 680 FAST) near infrared (NIR) fluorescence imaging agents to detect BBB breakdown and inflammation, respectively, we quantified brain and spinal cord changes in mice with relapsing-remitting PLP139-151-induced EAE and in response to tolerogenic therapy. RESULTS: FMT imaging and analysis techniques were carefully characterized and non-invasive imaging results corroborated by both ex vivo tissue imaging and comparison to clinical score results and histopathological analysis of CNS tissue. FMT imaging showed clear differences between control and diseased mice, and immune tolerance induction by antigen-coupled PLGA nanoparticles effectively blocked both disease induction and accumulation of imaging agents in the brain and spinal cord. CONCLUSIONS: Cat B 680 FAST and AngioSense 750EX offered the combination best able to detect disease in both the brain and spinal cord, as well as the downregulation of disease by antigen-specific tolerance. Non-invasive optical tomographic imaging thus offers a unique approach to monitoring neuroinflammatory disease and therapeutic intervention in living mice with EAE.

Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis.

Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.

Aerosol delivery of virus-like particles to the genital tract induces local and systemic antibody responses.

The induction of mucosal immune responses in the genital tract may be important for increasing the effectiveness of vaccines for sexually transmitted infections (STIs). In this study, we asked whether direct immunization of the mouse genital tract with a non-replicating virus-like particle (VLP)-based vaccine could induce local mucosal as well as systemic antibody responses. Using VLPs derived from two bacteriophages, Qß and PP7, and from a mammalian virus that normally infects the genital tract, human papillomavirus (HPV), we show that intravaginal aerosol administration of VLPs can induce high titer IgG and IgA antibodies in the female genital tract as well as IgG in the sera. Using a mouse model for HPV infection, we show that intravaginal immunization with either HPV type 16 VLPs or with PP7 bacteriophage VLPs displaying a peptide derived from the HPV minor capsid protein L2 could protect mice from genital infection with an HPV16 pseudovirus. These results provide a general method for inducing genital mucosal and systemic antibody responses using VLP-based immunogens.