Liquid foam improves potency and safety of gene therapy vectors.

Interest in gene therapy medicines is intensifying as the first wave of gene-correcting drugs is now reaching patient populations. However, efficacy and safety concerns, laborious manufacturing protocols, and the high cost of the therapeutics are still significant barriers in gene therapy. Here we describe liquid foam as a vehicle for gene delivery. We demonstrate that embedding gene therapy vectors (nonviral or viral) in a methylcellulose/xanthan gum-based foam formulation substantially boosts gene transfection efficiencies in situ, compared to liquid-based gene delivery. We further establish that our gene therapy foam is nontoxic and retained at the intended target tissue, thus minimizing both systemic exposure and targeting of irrelevant cell types. The foam can be applied locally or injected to fill body cavities so the vector is uniformly dispersed over a large surface area. Our technology may provide a safe, facile and broadly applicable option in a variety of clinical settings.

Biomaterial-based scaffolds for direct in situ programming of tumor-infiltrating T lymphocytes.

Adoptive cell therapy with tumor-infiltrating T cells (TILs) has generated exciting clinical trial results for the treatment of unresectable solid tumors. However, solid tumors remain difficult targets for adoptively transferred T cells, due in part to poor migration of TILs to the tumor, physical barriers to infiltration, and active suppression of TILs by the tumor. Furthermore, a highly skilled team is required to obtain tumor tissue, isolate and expand the TILs ex vivo, and reinfuse them into the patient, which drives up costs and limits patient access. Here, we describe a cell-free polymer implant designed to recruit, genetically reprogram and expand host T cells at tumor lesions in situ. Importantly, the scaffold can be fabricated on a large scale and is stable to lyophilization. Using a mouse breast cancer model, we show that the implants quickly and efficiently amass cancer-specific host lymphocytes at the tumor site in quantities sufficient to bring about long-term tumor regression. Given that surgical care is the mainstay of cancer treatment for many patients, this technology could be easily implemented in a clinical setting as an add-on to surgery for solid tumors. Furthermore, the approach could be broadened to recruit and genetically reprogram other therapeutically desirable host cells, such as macrophages, natural killer cells or dendritic cells, potentially boosting the antitumor effectiveness of the implant even more.

Development of a Smart Wireless Multisensor Platform for an Optogenetic Brain Implant.

Implantable cell replacement therapies promise to completely restore the function of neural structures, possibly changing how we currently perceive the onset of neurodegenerative diseases. One of the major clinical hurdles for the routine implementation of stem cell therapies is poor cell retention and survival, demanding the need to better understand these mechanisms while providing precise and scalable approaches to monitor these cell-based therapies in both pre-clinical and clinical scenarios. This poses significant multidisciplinary challenges regarding planning, defining the methodology and requirements, prototyping and different stages of testing. Aiming toward an optogenetic neural stem cell implant controlled by a smart wireless electronic frontend, we show how an iterative development methodology coupled with a modular design philosophy can mitigate some of these challenges. In this study, we present a miniaturized, wireless-controlled, modular multisensor platform with fully interfaced electronics featuring three different modules: an impedance analyzer, a potentiostat and an optical stimulator. We show the application of the platform for electrical impedance spectroscopy-based cell monitoring, optical stimulation to induce dopamine release from optogenetically modified neurons and a potentiostat for cyclic voltammetry and amperometric detection of dopamine release. The multisensor platform is designed to be used as an opto-electric headstage for future in vivo animal experiments.

Interventional Treatment of Symptomatic Vasospasm in the Setting of Traumatic Brain Injury: A Systematic Review of Reported Cases.

Traumatic subarachnoid hemorrhage (tSAH) is frequently comorbid with traumatic brain injury (TBI) and may induce secondary injury through vascular changes such as vasospasm and subsequent delayed cerebral ischemia (DCI). While aneurysmal SAH is well studied regarding vasospasm and DCI, less is known regarding tSAH and the prevalence of vasospasm and DCI, the consequences of vasospasm in this setting, when treatment is indicated, and which management strategies should be implemented. In this article, a systematic review of the literature that was conducted for cases of symptomatic vasospasm in patients with TBI is reported, association with tSAH is reported, risk factors for vasospasm and DCI are summarized, and commonalities in diagnosis and management are discussed. Clinical characteristics and treatment outcomes of 38 cases across 20 studies were identified in which patients with TBI with vasospasm underwent medical or endovascular management. Of the patients with data available for each category, the average age was 48.7 ± 20.3 years (n = 31), the Glasgow Coma Scale score at presentation was 10.6 ± 4.5 (n = 35), and 100% had tSAH (n = 29). Symptomatic vasospasm indicative of DCI was diagnosed on average at postinjury day 8.4 ± 3.0 days (n = 30). Of the patients, 56.6% (n = 30) had a new ischemic change associated with vasospasm confirming DCI. Treatment strategies are discussed, with 11 of 12 endovascularly treated and 19 of 26 medically treated patients surviving to discharge. tSAH is associated with vasospasm and DCI in moderate and severe TBI, and patients with clinical and radiographic evidence of symptomatic vasospasm and subsequent DCI may benefit from endovascular or medical management strategies.

Modular 3D printed platform for fluidically connected human brain organoid culture.

Brain organoid technology has transformed both basic and applied biomedical research and paved the way for novel insights into developmental processes and disease states of the human brain. While the use of brain organoids has been rapidly growing in the past decade, the accompanying bioengineering and biofabrication solutions have remained scarce. As a result, most brain organoid protocols still rely on commercially available tools and culturing platforms that had previously been established for different purposes, thus entailing suboptimal culturing conditions and excessive use of plasticware. To address these issues, we developed a 3D printing pipeline for the fabrication of tailor-made culturing platforms for fluidically connected but spatially separated brain organoid array culture. This all-in-one platform allows all culturing steps-from cellular aggregation, spheroid growth, hydrogel embedding, and organoid maturation-to be performed in a single well plate without the need for organoid manipulation or transfer. Importantly, the approach relies on accessible materials and widely available 3D printing equipment. Furthermore, the developed design principles are modular and highly customizable. As such, we believe that the presented technology can be easily adapted by other research groups and fuel further development of culturing tools and platforms for brain organoids and other 3D cellular systems.

Novel Conformation-Dependent Tau Antibodies Are Modulated by Adjacent Phosphorylation Sites.

Tau proteins within the adult central nervous system (CNS) are found to be abnormally aggregated into heterogeneous filaments in neurodegenerative diseases, termed tauopathies. These tau inclusions are pathological hallmarks of Alzheimer's disease (AD), Pick's disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). The neuropathological hallmarks of these diseases burden several cell types within the CNS, and have also been shown to be abundantly phosphorylated. The mechanism(s) by which tau aggregates in the CNS is not fully known, but it is hypothesized that hyperphosphorylated tau may precede and further promote filament formation, leading to the production of these pathological inclusions. In the studies herein, we generated and thoroughly characterized two novel conformation-dependent tau monoclonal antibodies that bind to residues Pro218-Glu222, but are sensitive to denaturing conditions and highly modulated by adjacent downstream phosphorylation sites. These epitopes are present in the neuropathological hallmarks of several tauopathies, including AD, PiD, CBD, and PSP. These novel antibodies will further enable investigation of tau-dependent pathological inclusion formation and enhance our understanding of the phosphorylation signatures within tauopathies with the possibility of new biomarker developments.

Updated trends in percutaneous renal arteriography among radiologists and other specialties.

PURPOSE: Prior studies have demonstrated an overall decline in percutaneous renal artery angioplasty with and without stenting from 1988 to 2009. We evaluated the recent utilization trends in percutaneous renal arteriography (PTRA) among radiologists and non-radiologist providers from 2010 to 2018. METHODS: Data from the 2010-2018 nationwide Medicare Part B fee-for-service database were used to tabulate case volumes for PTRA. Annual utilization rates per 10,000 Medicare beneficiaries were calculated and aggregated based on physician specialty: radiologists, cardiologists, vascular surgeons, general surgeons, or others. RESULTS: From 2010 to 2018, the overall utilization rate of PTRA markedly declined (-72% change; from 15.5 to 4.3 cases per 10,000 Medicare beneficiaries). Proportionally, the cardiologist share of PTRA saw the greatest decline, falling from 74% market share in 2010 (11.4/15.5 cases) to only 36% market share in 2018 (1.6/4.3 cases). The market share of PTRA performed by radiologists grew from 12% market share in 2010 (1.9/15.5 cases) to 28% in 2018 (1.2/4.3 cases); despite this, the absolute number of PTRA performed by radiologists saw a smaller decline over this period (-34%; 1.9 to 1.2 cases). CONCLUSION: The total utilization rates of PTRA in the Medicare population has continued to decline from 2010 to 2018, likely due to clinical trials suggesting limited efficacy of angioplasty and stenting in the treatment of renovascular hypertension and other factors such as declining reimbursement. The overall and per-specialty rates continue to decline, reflecting an overarching trend away from procedural management of renovascular hypertension.

Dual-Energy CT in Cardiothoracic Imaging: Current Developments.

This article describes the technical principles and clinical applications of dual-energy computed tomography (DECT) in the context of cardiothoracic imaging with a focus on current developments and techniques. Since the introduction of DECT, different vendors developed distinct hard and software approaches for generating multi-energy datasets and multiple DECT applications that were developed and clinically investigated for different fields of interest. Benefits for various clinical settings, such as oncology, trauma and emergency radiology, as well as musculoskeletal and cardiovascular imaging, were recently reported in the literature. State-of-the-art applications, such as virtual monoenergetic imaging (VMI), material decomposition, perfused blood volume imaging, virtual non-contrast imaging (VNC), plaque removal, and virtual non-calcium (VNCa) imaging, can significantly improve cardiothoracic CT image workflows and have a high potential for improvement of diagnostic accuracy and patient safety.

Omnidirectional leaky opto-electrical fiber for optogenetic control of neurons in cell replacement therapy.

The pathophysiological progress of Parkinson's disease leads through degeneration of dopaminergic neurons in the substantia nigra to complete cell death and lack of dopamine in the striatum where it modulates motor functions. Transplantation of dopaminergic stem cell-derived neurons is a possible therapy to restore dopamine levels. We have previously presented multifunctional pyrolytic carbon coated leaky optoelectrical fibers (LOEFs) with laser ablated micro-optical windows (µOWs) as carriers for channelrhodopsin-2 modified optogenetically active neurons for light-induced on-demand dopamine release and amperometric real-time detection. To increase the dopamine release by stimulating a larger neuronal population with light, we present here a novel approach to generate µOWs through laser ablation around the entire circumference of optical fibers to obtain Omni-LOEFs. Cyclic voltammetric characterization of the pyrolytic carbon showed that despite the increased number of µOWs, the electrochemical properties were not deteriorated. Finally, we demonstrate that the current recorded during real-time detection of dopamine upon light-induced stimulation of neurons differentiated on Omni-LOEFs is significantly higher compared to recordings from the same number of cells seeded on LOEFs with µOWs only on one side. Moreover, by varying the cell seeding density, we show that the recorded current is proportional to the dimension of the cell population.

Retrospective Multicenter Study on Outcome Measurement for Dyskinesia Improvement in Parkinson's Disease Patients with Pallidal and Subthalamic Nucleus Deep Brain Stimulation.

Deep brain stimulation (DBS) is an effective treatment for dyskinesia in patients with Parkinson's disease (PD), among which the therapeutic targets commonly used include the subthalamic nucleus (STN) and the globus pallidus internus (GPi). Levodopa-induced dyskinesia (LID) is one of the common motor complications arising in PD patients on chronic treatment with levodopa. In this article, we retrospectively evaluated the outcomes of LID with the Unified Dyskinesia Rating Scale (UDysRS) in patients who underwent DBS in multiple centers with a GPi or an STN target. Meanwhile, the Med off MDS-Unified Parkinson's Disease Rating Scale (MDS-UPDRS-Ⅲ) and the levodopa equivalent daily dose (LEDD) were also observed as secondary indicators. PD patients with a GPi target showed a more significant improvement in the UDysRS compared with an STN target (92.9 ± 16.7% vs. 66.0 ± 33.6%, p < 0.0001). Both the GPi and the STN showed similar improvement in Med off UPDRS-III scores (49.8 ± 22.6% vs. 52.3 ± 29.5%, p = 0.5458). However, the LEDD was obviously reduced with the STN target compared with the GPi target (44.6 ± 28.1% vs. 12.2 ± 45.8%, p = 0.006).

Pathogenic tau recruits wild-type tau into brain inclusions and induces gut degeneration in transgenic SPAM mice.

Pathological tau inclusions are neuropathologic hallmarks of many neurodegenerative diseases. We generated and characterized a transgenic mouse model expressing pathogenic human tau with S320F and P301S aggregating mutations (SPAM) at transgene levels below endogenous mouse tau protein levels. This mouse model develops a predictable temporal progression of tau pathology in the brain with biochemical and ultrastructural properties akin to authentic tau inclusions. Surprisingly, pathogenic human tau extensively recruited endogenous mouse tau into insoluble aggregates. Despite the early onset and rapid progressive nature of tau pathology, major neuroinflammatory and transcriptional changes were only detectable at later time points. Moreover, tau SPAM mice are the first model to develop loss of enteric neurons due to tau accumulation resulting in a lethal phenotype. With moderate transgene expression, rapidly progressing tau pathology, and a highly predictable lethal phenotype, the tau SPAM model reveals new associations of tau neurotoxicity in the brain and intestinal tract.

A bioinformatics pipeline for estimating mitochondrial DNA copy number and heteroplasmy levels from whole genome sequencing data.

Mitochondrial diseases are a heterogeneous group of disorders that can be caused by mutations in the nuclear or mitochondrial genome. Mitochondrial DNA (mtDNA) variants may exist in a state of heteroplasmy, where a percentage of DNA molecules harbor a variant, or homoplasmy, where all DNA molecules have the same variant. The relative quantity of mtDNA in a cell, or copy number (mtDNA-CN), is associated with mitochondrial function, human disease, and mortality. To facilitate accurate identification of heteroplasmy and quantify mtDNA-CN, we built a bioinformatics pipeline that takes whole genome sequencing data and outputs mitochondrial variants, and mtDNA-CN. We incorporate variant annotations to facilitate determination of variant significance. Our pipeline yields uniform coverage by remapping to a circularized chrM and by recovering reads falsely mapped to nuclear-encoded mitochondrial sequences. Notably, we construct a consensus chrM sequence for each sample and recall heteroplasmy against the sample's unique mitochondrial genome. We observe an approximately 3-fold increased association with age for heteroplasmic variants in non-homopolymer regions and, are better able to capture genetic variation in the D-loop of chrM compared to existing software. Our bioinformatics pipeline more accurately captures features of mitochondrial genetics than existing pipelines that are important in understanding how mitochondrial dysfunction contributes to disease.

Vertebral stabilisation for thoracolumbar vertebral instability associated with cranial and caudal articular process anomalies in pugs: Seven cases (2010-2019).

OBJECTIVES: To describe the diagnostic findings, surgical technique and outcomes in seven pugs with thoracolumbar vertebral instability due to articular process anomalies. MATERIALS AND METHODS: Records (2010 to 2019) of pugs with thoracolumbar vertebral instability associated with articular process anomalies that underwent decompressive laminectomy and vertebral stabilisation were reviewed. Data on preoperative and postoperative neurologic status, diagnostic findings, surgical techniques and outcomes were retrieved. RESULTS: Seven dogs were presented with ambulatory or non-ambulatory paraparesis. Caudal articular process anomalies (three dogs) and concomitant cranial and caudal articular process anomalies (four dogs) were noted. Myelography (six dogs) or magnetic resonance imaging (one dog) showed none to severe spinal cord compression. Dynamic myelography in six dogs demonstrated nine distinct sites of spinal cord dimension reduction positioned in extension and/or flexion (mean reduction: 16.0%, range: 8.5 to 24.0%). These dynamic compressions were located at sites with articular process anomalies (seven sites) and sites with no articular process anomalies (two sites). Vertebral instability was confirmed by intraoperative spinal manipulation in all dogs. All dogs remained ambulatory with improved (five dogs) or static (two dogs) neurological deficits at the last follow-up (median: 16 months; range: 1.5 to 66 months). CLINICAL SIGNIFICANCE: Dynamic myelography and/or intraoperative spinal manipulation demonstrated vertebral instabilities at sites with or without articular process anomalies on imaging. Decompressive laminectomy with vertebral stabilisation resulted in long-term neurological improvement in most dogs.

Multimodality Imaging of Pleuropulmonary Blastoma: Pearls, Pitfalls, and Differential Diagnosis.

Pleuropulmonary blastomas are rare, potentially aggressive embryonal cancers of the lung parenchyma and pleural surfaces that account for 0.25%-0.5% of primary pulmonary malignancies in children. Pleuropulmonary blastomas are classified as cystic (type I), mixed cystic and solid (type II), and solid (type III). Pleuropulmonary blastoma occurs in the same age group (0-6 years) as other more common solid tumors such as neuroblastoma and Wilms tumor. Differential diagnosis includes metastasis from Wilms tumor and macrocystic congenital pulmonary airway malformation (CPAM). A key pathologic and genetic discriminator is the DICER1 germline mutation found in patients with pleuropulmonary blastoma. Imaging, histopathologic, and clinical data are important to use in conjunction in order to determine the diagnosis and risk stratification of pleuropulmonary blastomas. Survival varies from poor to good, depending on type. However, the spectrum of pleuropulmonary blastoma is insufficiently understood due to the variable presentation of this rare disease. We present a current review of the literature regarding pleuropulmonary blastomas in this article.

BiTE secretion from in situ-programmed myeloid cells results in tumor-retained pharmacology.

Bispecific T-Cell Engagers (BiTEs) are effective at inducing remission in hematologic cancers, but their use in solid tumors has been challenging due to their extreme potency and on-target, off-tumor toxicities in healthy tissue. Their deployment against solid tumors is further complicated by insufficient drug penetration, a hostile tumor microenvironment, and immune escape. To address these challenges, we developed targeted nanocarriers that can deliver in vitro-transcribed mRNA encoding BiTEs to host myeloid cells - a cell type that is actively recruited into the tumor microenvironment. We demonstrate in an immunocompetent mouse model of ovarian cancer, that infusion of these nanoparticles directs BiTE expression to tumor sites, which reshapes the microenvironment from suppressive to permissive and triggers disease regression without systemic toxicity. In contrast, conventional injections of recombinant BiTE protein at doses required to achieve anti-tumor activity, induced systemic inflammatory responses and severe tissue damage in all treated animals. Implemented in the clinic, this in situ gene therapy could enable physicians - with a single therapeutic - to safely target tumor antigen that would otherwise not be druggable due to the risks of on-target toxicity and, at the same time, reset the tumor milieu to boost key mediators of antitumor immune responses.

Convalescent Plasma Therapy in Critically Ill COVID-19 Patients: An Open Label Trial.

OBJECTIVES: The novel severe acute respiratory syndrome coronavirus 2 pandemic continues to spread globally without an effective treatment. In search of the cure, convalescent plasma (CP) containing protective antibodies from survivors of coronavirus disease 2019 (COVID-19) infection has shown potential benefit in a non-intensive care unit setting. We sought to evaluate the effectiveness of CP therapy for patients with COVID-19 on mechanical ventilation (MV) and/or acute respiratory distress syndrome (ARDS). METHODS: We conducted an open-label trial in a single center, Royal Hospital, in Oman. The study was conducted from 17 April to 20 June 2020. The trial included 94 participants with laboratory-confirmed COVID-19. The primary outcomes included extubation rates, discharges from the hospital and overall mortality, while secondary outcomes were the length of stay and improvement in respiratory and laboratory parameters. Analyses were performed using univariate statistics. RESULTS: The overall mean age of the cohort was 50.0±15.0 years, and 90.4% (n = 85) were males. A total of 77.7% (n = 73) of patients received CP. Those on CP were associated with a higher extubation rate (35.6% vs. 76.2%; p < 0.001), higher extubation/home discharges rate (64.4% vs. 23.8%; p =0.001), and tendency towards lower overall mortality (19.2% vs. 28.6%; p =0.354; study power = 11.0%) when compared to COVID-19 patients that did not receive CP. CONCLUSIONS: CP was associated with higher extubation/home discharges and a tendency towards lower overall mortality when compared to those that did not receive CP in COVID-19 patients on MV or in those with ARDS. Further studies are warranted to corroborate our findings.

Genetic in situ engineering of myeloid regulatory cells controls inflammation in autoimmunity.

The ability of myeloid regulatory cells (MRCs) to control immune responses and to promote tolerance has prompted enormous interest in exploiting them therapeutically to treat inflammation, autoimmunity, or to improve outcomes in transplantation. While immunomodulatory small-molecule compounds and antibodies have provided relief for some patients, the dosing entails high systemic drug exposures and thus increased risk of off-target adverse effects. More recently, MRC-based cell-therapy products have entered clinical testing for tolerance induction. However, the elaborate and expensive protocols currently required to manufacture engineered MRCs ex vivo put this approach beyond the reach of many patients who might benefit. A solution could be to directly program MRCs in vivo. Here we describe a targeted nanocarrier that delivers in vitro-transcribed mRNA encoding a key anti-inflammatory mediator. We demonstrate in models of systemic lupus erythematosus that infusions of nanoparticles formulated with mRNA encoding glucocorticoid-induced leucine zipper (GILZ) effectively control the disease. We further establish that these nanoreagents are safe for repeated dosing. Implemented in the clinic, this new therapy could enable physicians to treat autoimmune disease while avoiding systemic treatments that disrupt immune homeostasis.

Hot punching for loading of biodegradable microcontainers with budesonide-Soluplus film.

Micro-reservoir based drug delivery systems have the potential to provide targeted drug release locally in the intestine, i.e. at the inflamed areas of the intestine of patients with inflammatory bowel disease (IBD). In this study, microcontainers with a diameter of 300 µm and a height of 100 µm, asymmetrical geometry and the possibility to provide unidirectional release, are fabricated in the biodegradable polymer poly-ɛ-caprolactone (PCL) using hot punching. As a first step towards local treatment of IBD, a novel method for loading of microcontainers with the corticosteroid budesonide is developed. For this purpose, a budesonide-Soluplus drug-polymer film is prepared by spin coating and loaded into the microcontainer reservoirs using hot punching. The processing parameters are optimized to achieve a complete loading of a large number of containers in a single step. A poly(lactic-co-glycolic acid) (PLGA) 50:50 lid is subsequently applied by spray coating. Solid-state characterization indicates that the drug is in an amorphous state in the drug-polymer films and the in vitro drug release profile showed a 68% release over 10 h. The results demonstrate that hot punching can be employed both as a production and loading method for PCL microcontainers with the perspective of local treatment of IBD.